Use of protein biomolecular targets in the treatment and visualization of brain tumors

ABSTRACT

The present invention relates to the use of proteins which are differentially expressed in primary brain tumor tissues, as compared to normal brain tissues, as biomolecular targets for brain tumor treatment therapies. Specifically, the present invention relates to the use of immunotherapeutic and immunoimaging agents which specifically bind to one or more of human proteins angiopoietin related protein 2 (ARP-2,) secreted protein acidic, rich in cysteine (SPARC,) c-met proto-oncogene (C-MET,) brevican (BEHAB,) CD-44 antigen (CD-44,) tetraspanin 3 (TSPN3,) pleiotrophin (PTN,) osteopontin (OPN,) vasoactive intestinal peptide receptor-2 (VIPR-2,) and receptor protein tyrosine phosphatase zeta (PTPζ) for the treatment and visualization of brain tumors in patients. The present invention also provides compounds and pharmaceutically acceptable compositions for administration in the methods of the invention. The present invention also provides novel splice variants of protein PTPζ, PTPζ SM1 and PTPζ SM2. Nucleic acid probes specific for the spliced mRNA encoding these variants and affinity reagents specific for the novel proteins are also provided.

FIELD OF USE

[0001] The present invention relates to the use of proteins which aredifferentially expressed in primary brain tumor tissues, as compared tonormal brain tissues, as biomolecular targets for brain tumor treatmenttherapies. Specifically, the present invention relates to the use ofimmunotherapeutic and immunoimaging agents which specifically bind toone or more of angiopoietin related protein 2 (ARP-2,) secreted proteinacidic, rich in cysteine (SPARC,) c-met proto-oncogene (C-MET,) brevican(BEHAB,) CD-44 antigen (CD-44,) tetraspanin 3 (TSPN3,) pleiotrophin(PTN,) osteopontin (OPN,) vasoactive intestinal peptide receptor-2(VIPR-2,) and receptor protein tyrosine phosphatase zeta (PTPζ) for thetreatment and visualization of brain tumors in patients. The presentinvention also provides compounds and pharmaceutically acceptablecompositions for administration.

BACKGROUND OF THE INVENTION

[0002] Brain Tumor Biology and Etiology

[0003] Brain tumors are considered to have one of the least favorableprognoses for long term survival: the average life expectancy of anindividual diagnosed with a central nervous system (CNS) tumor is justeight to twelve months. Several unique characteristics of both the brainand its particular types of neoplastic cells create daunting challengesfor the complete treatment and management of brain tumors. Among theseare 1) the physical characteristics of the intracranial space, 2) therelative biological isolation of the brain from the rest of the body, 3)the relatively essential and irreplaceable nature of the organ mass, and4) the unique nature of brain tumor cells.

[0004] First and foremost, the intracranial space and physical layout ofthe brain create significant obstacles to treatment and recovery. Thebrain is made of, primarily, astrocytes (which make up the majority ofthe brain mass, and serve as a scaffold and support for the neurons),neurons (which carry the actual electrical impulses of the nervoussystem), and a minor contingent of other cells such as insulatingoligodendrocytes (which produce myelin). These cell types give rise toprimary brain tumors (e.g., astrocytomas, neuroblastomas, glioblastomas,oligodendrogliomas, etc.) Although the World Health Organization hasrecently established standard guidelines, the nomenclature for braintumors is somewhat imprecise, and the terms astrocytoma and glioblastomaare often used broadly. The brain is encased in the relatively rigidshell of the skull, and is cushioned by the cerebrospinal fluid, muchlike a fetus in the womb. Because of the relatively small volume of theskull cavity, minor changes in the volume of tissue in the brain candramatically increase intracranial pressure, causing damage to theentire organ (i.e., “water on the brain”). Thus, even small tumors canhave a profound and adverse affect on the brain's function. In contrast,tumors in the relatively distensible abdomen may reach several pounds insize before the patient experiences adverse symptoms. The crampedphysical location of the cranium also makes surgery and treatment of thebrain a difficult and delicate procedure. However, because of thedangers of increased intracranial pressure from the tumor, surgery isoften the first strategy of attack in treating brain tumors.

[0005] In addition to its physical isolation, the brain is chemicallyand biologically isolated from the rest of the body by the so-called“Blood-Brain-Barrier” (or BBB). This physiological phenomenon arisesbecause of the “tightness” of the epithelial cell junctions in thelining of the blood vessels in the brain. Although nutrients, which areactively transported across the cell lining, may reach the brain, othermolecules from the bloodstream are excluded. This prevents toxins,viruses, and other potentially dangerous molecules from entering thebrain cavity. However, it also prevents therapeutic molecules, includingmany chemotherapeutic agents that are useful in other types of tumors,from crossing into the brain. Thus, many therapies directed at the brainmust be delivered directly into the brain cavity (e.g., by an Ommayareservoir), or administered in elevated dosages to ensure the diffusionof an effective amount across the BBB.

[0006] With the difficulties of administering chemotherapies to thebrain, radiotherapy approaches have also been attempted. However, theamount of radiation necessary to completely destroy potentialtumor-producing cells also produce unacceptable losses of healthy braintissue. The retention of patient cognitive function while eliminatingthe tumor mass is another challenge to brain tumor treatment. Neoplasticbrain cells are often pervasive, and travel throughout the entire brainmass. Thus, it is impossible to define a true “tumor margin,” unlike,for example, in lung or bladder cancers. Unlike reproductive (ovarian,uterine, testicular, prostate, etc.), breast, kidney, or lung cancers,the entire organ, or even significant portions, cannot be removed toprevent the growth of new tumors. In addition, brain tumors are veryheterogeneous, with different cell doubling times, treatmentresistances, and other biochemical idiosyncrasies between the variouscell populations that make up the tumor. This pervasive and variablenature greatly adds to the difficulty of treating brain tumors whilepreserving the health and function of normal brain tissue.

[0007] Although current surgical methods offer considerably betterpost-operative life for patients, the current combination therapymethods (surgery, low-dosage radiation, and chemotherapy) have onlyimproved the life expectancy of patients by one month, as compared tothe methods of 30 years ago. Without effective agents to prevent thegrowth of brain tumor cells that are present outside the main tumormass, the prognosis for these patients cannot be significantly improved.Although some immuno-affinity agents have been proposed and tested forthe treatment of brain tumors, see, e.g., the tenascin-targeting agentsdescribed in U.S. Pat. No. 5,624,659, these agents have not provensufficient for the treatment of brain tumors. Thus, therapeutic agentswhich are directed towards new molecular targets, and are capable ofspecifically targeting and killing brain tumor cells, are urgentlyneeded for the treatment of brain tumors.

[0008] ARP-2 (Angiopoeitin Related Protein-2, Angiopoeitin Like-2[ANGPTL-2])

[0009] Angiopoeitin related protein-2 (ARP-2), is related to theangiopoeitin family of proteins, that includes Ang-1 and Ang-2. Likemembers of the angiopoeitin family, ARP-2 contains a coiled-coil domainin the amino terminal portion and a fibrinogen-like domain in thecarboxyl terminal portion. However, ARP-2 has a low homology with Ang-1and Ang-2 and unlike Ang-1 and Ang-2, ARP-2 does not bind to the Tie-2receptor, nor does ARP-2 bind to the closely related Tie-1 receptor.Hence, ARP-2 is believed to be part of a newly identified family ofproteins termed angiopocitin related proteins. Like the angiopoeitins,ARP-2 is a member of the fibrinogen superfamily, which also includes thefibrinogens and lectins.

[0010] ARP-2 is a glycosylated, secretory protein that induces sproutingin endothelial cells, most likely through autocrine or paracrinesignaling, and it is preferentially expressed in the blood vessels andmuscle cells. Hence, ARP-2 mediates the differentiated state ofendothelial cells or for vascular remodeling and development. ARP-2 hasnot heretofore been associated with brain tumors.

[0011] SPARC (Secreted Protein, Aacidic, Cysteine-Rich; Osteonectin;Basement Membrane Protein (bm) 40)

[0012] Secreted protein acidic and rich in cysteine, SPARC or BM-40, isa member of the counter-adhesive family of proteins. It is adevelopmentally regulated, secreted glycoprotein expressed in fetalastrocytes, particularity during tissue remodeling, vesselmorphogenesis, and in response to stress. It has been hypothesized thatSPARC may affect cell migration and vascular morphogenesis either bydirectly interacting with extracellular matrix (ECM) proteins (such ascollagens I, III, IV and V) or by initiating a receptor mediatedsignaling event that induces changes in cytoplasmic componentsassociated with focal adhesions. SPARC has been found to bind directlyto vitronectin, a multifunctional adhesive protein that is a componentof the brain vascular basement membranes.

[0013] SPARC may indirectly affect cell migration and motility byregulating the expression of matrix metallo-proteases and by modulatingthe expression of other proteolytic enzymes (such as collagenase) thatdegrade the ECM. Increased SPARC expression has also been observed intwo forms of low-grade malignant gliomas, in all grades of humanastrocytic tumors, and in tumor cells invading adjacent brain at thetumor/brain interface. Hence, SPARC may be an astrocytoma invasionrelated gene that functions in connection with vitronectin to balancethe modulation of cellular adhesion to the ECM and it may promotediffuse tumor cell infiltration into adjacent brain by affecting bothtumor and endothelial cell-ECM interactions.

[0014] Because SPARC is also found in bone, dentine, and many normal andneoplastic human soft tissues it may also play a regulatory function inthe control of such diverse processes as bone mineralization, cellshape, tissue remodeling or repair, cell migration, proliferation, anddifferentiation. SPARC is also synthesized, stored, and secreted byhuman blood platelets, binds to plasminogen, and enhances tissueplasminogen activator conversion of plasminogen to plasmin.

[0015] c-MET (Met Proto-Oncogene Tyrosine Kinase, Hepatocyte GrowthFactor Receptor [HGFR])

[0016] c-MET is a member of the Hepatocyte Growth Factor Receptor (HGFR)family and a heterodimeric cellular receptor for Hepatocyte GrowthFactor (HGF). c-MET contains a disulfide-linked α-chain of 50-kDa (whichis located in the extracellular domain,) a 145-kDa β-chain (whichincludes an extracellular region,) a transmembrane spanning domain, andan intracellular tyrosine kinase domain that can be activated byautophosphorylation. Hence, HGFR is a subset of the proteintyrosine-kinase family of membrane-spanning, cell surface receptors.

[0017] The receptor-ligand pair, c-MET and HGF, function as a growthfactor, regulating cell growth, migration, and morphogenesis, and hence,may play a role in neoplastic formation and metastasis. Upon HGF ormacrophage stimulating protein (MSP) binding, the c-MET protein receptorgoes through a conformational change wherein the intracellular tyrosineresidues of the β subunit become phosphorylated at residue 1235, and asecond messenger signal cascade is induced. This change activatesc-MET's intracellular receptor kinase activity, which is important tothe growth and differentiation of epithelial cells in normal andmalignant tissues. c-MET has been identified in both normal brain and onglial tumors, and is thought to be determinant in the pathologicalprocesses of various malignancies. For instance, detailed studies haveshown that glioblastoma multiforme (GBM), a highly malignant brain tumorof astrocytic origin, expresses c-MET, and this research suggests a rolein tumor progression.

[0018] BEHAB (Brain-Enriched Hyaluronan Binding Protein, Brevican)

[0019] BEHAB is a brain-specific, extracellular matrix protein, that isa member of the chondroitin sulfate proteoglycan (CSPG) family. BEHAB isexpressed only in the CNS. Although its function is unclear, BEHAB isreported to bind to HA at the N-terminus, lectins at the C-terminus, andmay mediate binding of other ECM components like tenascin. This suggeststhat BEHAB may play a role in cell-cell and cell-matrix interactionsthereby maintaining the extracellular environment of the brain. It hasbeen reported that the highest levels of expression of BEHAB is duringbrain development and at times and places where glial cells are highlymotile, as in cases of brain injury or trauma. BEHAB expression is alsounregulated in primary gliomas of the central nervous system, but not intumors of non-glial origin. In surgical samples of human gliomas(including astrocytoma, oligodendroglioma, and glioblastoma tumors),BEHAB expression is consistently and dramatically increased over thelevel of expression in the normal brain. Hence, BEHAB expressioncorrelates with an invasive phenotype that promotes gliogenesis bycontributing to cell movement through the ECM.

[0020] CD-44 Antigen

[0021] CD-44 is a single-path, type I transmembrane protein withextracellular domains that are flexibly linked to the transmembranesegment. CD-44 is a member of the cartilage link protein family andbelongs to the hyaloadherin or link protein superfamily (LPSF). As othermembers of the LPS family, CD-44 can be extensively glycosylated and istypically decorated with glycosaminoglycans (e.g., chondroitin, heparin,and keratin sulfate). The genomic structure of CD-44 consists of 21exons, at least 11 of which can be variably spliced (v1 -v10), that arelocated in the membrane-proximal extracellular region. Alternativesplicing of these exons give rise to a variety of CD-44 isoforms (atleast 30 different isoforms have been characterized to date) that arewidely distributed and expressed in a cell-specific manner. Among themost frequently occurring isoforms are CD-44H, expressed onhematopoietic cells, and CD-44E, expressed in epithelial cells. CD-44(H)has also been found to be expressed in lymphocytes, macrophages,erythrocytes, fibroblasts, epithelial and endothelial cells, andneurons. It is the predominant isoform in normal brain andneuroectoderm-derived tumors and is expressed on both normal astrocytesand oligodendrocytes as well on neoplastic astrocytes and glioblastomas.

[0022] The family of CD-44 proteins has been implicated in lymphocyteactivation and homing, endothelial migration, and tumor cell metastasis.CD-44 is believed to be the major receptor for Hyaluronic acid (HA).CD-44/HA interactions underlie a wide spectrum of functions in embryonicmorphogenesis and organogenesis, hematopoeisis, lymphocyte homing. CD-44also mediates the attachment of glioma cells to chondroitin sulfate,types I and IV collagen, fibronectin laminin, vitronectin and Martrigel.This suggest that CD-44 may play a role in cell-cell and cell-matrixinteractions, affecting the extracellular environment of the brain.Because HA is a major component of the brain ECM, and CD-44 is one ofthe principal cellular receptors of HA, CD-44 expression coincides withbrain tumor growth and invasiveness.

[0023] PTN (Pleiotrophin, Heparin Binding Growth Factor 8, NeuriteGrowth-Promoting Factor 1)

[0024] Pleiotrophin or PTN, is a platelet-derived, growth factorinducible, member of the pleiotrophin family of proteins that includesmidkine and retinoic acid-induced heparin-binding protein. It is adevelopmentally regulated, secreted cytokine that stimulatesmitogenesis, angiogenesis, and neurite and glial process outgrowthguidance activities. During development PTN is expressed in the brain,intestine, muscle, skin, heart, lung and kidney. In the adult, PTN isfound primarily in the brain in association with axonal tracts duringactive mitogenesis and may therefore play an important role in thedevelopment and maintenance of the nervous system. It has been found tobind heparin, heparin sulfate proteoglycans, the extracellular matrix,and is also a natural ligand for receptor protein tyrosine phosphatase(RPTP), signaling through ligand dependant receptor inactivation ofRPTP. Receptor mediated endocytosis occurs following PTN binding and maybe disrupted by heparin.

[0025] PTN has also been found to have oncogenic properties, inducingmalignant transformation and tumor growth and progression. It has beendescribed as a proto-oncogene that is expressed in many human tumors andcell lines derived from human tumors. PTN is a mitogen for fibroblasts,epithelial and endothelial cells, stimulates plasminogen-activatorproduction, can induce tube formation, and therefore can serve as atumor angiogenesis factor.

[0026] OPN (Osteopontin, Secreted Phosphoprotein 1, Bone Sialoprotein-1)

[0027] Osteopontin or OPN, is a member of the osteopontin family. It isa glycosylated sialoprotein that is heavily phosphorylated and expressedin a variety of cells including bone, kidney, placenta, nerve cells andmacrophages, as well as T lymphocytes, epidermal and bone cells. OPN isa part of the mineralized bone matrix and may play a role in boneresorption, by facilitating the attachment of osteoclasts to the bonesurface, and may be functionally important as an adhesive andchemotactic molecule for vascular cells. OPN is a secreted protein thatbinds tightly to hydroxyapatite, and hence, is important to cell matrixinteractions. It has been observed to interact with the CD-44 homingreceptor to physiologically induce macrophage chemotaxis, which may be amechanism utilized by metastatic brain tumors in the process ofdissemination.

[0028] OPN has been observed in the microvasculature of glioblastomasassociated with VEGF expression and OPN mRNA has been found to beoverexpressed in high grade and metastatic brain tumors. Hence, OPNexpression correlates with the malignancy grade of gliomas.

[0029] VIPR-2 (Vasoactive Intestinal Peptide Receptor-2)

[0030] Vasoactive intestinal polypeptide receptor II (VIPR-2), VPAC-2,is a member of the G-protein receptor family, which includes suchmembers as the calcitonin, parathyroid hormone, secretin, glucagon andVIP-1 receptors. VIPR-2 is a seven-transmembrane spanning Gprotein-coupled receptor that responds to VIP by stimulating cAMPproduction. VIPR-2 is found in the brain as well as peripheral tissuessuch as the pancreas, skeletal muscle, heart, lung, kidneys, stomach,adipocytes and the liver, and in various cells of the immune system. Inthe brain, VIPR-2 functions as a neuroendocrine hormone andneurotransmitter receptor, and is found in the thalamus, hippocampus,suprachiasmatic nucleus and hypothalamus.

[0031] VIPR-2 is encoded by a nucleotide sequence of approximately 2.8kb, which codes for a 438 amino acid sequence of approximately 48-64kDa. The receptor-ligand pair, VIPR-2 and VIP, have various functionsdependent upon the tissue where in they are located. VIP is alate-developing, 28 amino acid peptide that, along with its receptor, iswidely distributed throughout the peripheral body, and plays a role incardiovascular, reproductive, pulmonary, immune and gastrointestinalsystems, to effect vasodilatation, bronchodilation, immunosuppression,hormonal secretion, and increased gastric motility. However, thecerebral cortex has one of the highest reported concentrations of VIP,localized to intrinsic neurons throughout all neocortical regions. Inthe brain, VIP and its receptor, have behavioral, electrophysiological,secretory, metabolic, vascular, and mitogenic effects. For instance, thereceptor-ligand pair play a role in cortical differentiation, therelaying of sensory information to the cortex, and the regulation ofmorphogenic events by the release of diffusible signals from glialcells. VIPR-2 and VIP also play a role in the growth and differentiationof neuroblastomas.

[0032] TSPAN3 (Tetraspanin 3, Tetraspanin TM-4A)

[0033] The Tetraspanin superfamily, is a family of approximately 20integral membrane proteins that are broadly expressed in most humantissues including neural and bone marrow derived tissues. The familyshares a common motif that includes four putative transmembrane domains(TM1-4), a small extracellular domain (EC1) of 20-27 amino acids, and alarger extracellular domain (EC2) between TMS3 and TMS4 of 70-130 aminoacids. Two conserved features of tetraspanins are critical to theirstructure and function. First, charged residues are present in or nearthe TM domains, second, a cluster of cysteine residues is in theputative EC2 domain. Most of the tetraspanins are modified byN-glycosylation.

[0034] Many Tetraspanin proteins affect the regulation of cellularproliferation, motility, differentiation, development. In some cells,Tetraspanins may act as adapters in ultimeric complexes that link plasmamembrane proteins, like integrins, into signaling complexes with othersignaling molecules (e.g., phosphatidylinositol 4-kinase) at the plasmamembrane and play a role in integrin-mediated cell migration, metastasisand tumor cell invasion. A number of tetraspanins have also beendiscovered as tumor-associated proteins, including C-029, PETA-3/SFA-1,and SAS, which is amplified in a subset of sarcomas. Of the variousTM4SF proteins, CD9, CD63, CD81, CD82, and CD151 are the most widelydistributed. CD9 is expressed on 90% of non-T cell acute lymphoblasticleukemia cells and on 50% of chronic lymphocytic and acute myeloblasticleukemias. CD63 is also expressed in early stage melanomas.

[0035] Protein Tyrosine Phosphatase Receptor Zeta (PTPζ)

[0036] Vital cellular functions, such as cell proliferation and signaltransduction, are regulated in part by the balance between theactivities of protein kinases and protein phosphatases. Theseprotein-modifying enzymes add or remove a phosphate group from serine,threonine, or tyrosine residues in specific proteins. Some tyrosinekinases (PTK's) and phosphatases (PTPase's) have been theorized to havea role in some types of oncogenesis, which is thought to result from animbalance in their activities. There are two classes of PTPasemolecules: low molecular weight proteins with a single conservedphosphatase domain such as T-cell protein-tyrosine phosphatase (PTPT;MIM 176887), and high molecular weight receptor-linked PTPases with twotandemly repeated and conserved phosphatase domains separated by 56 to57 amino acids. Examples of this latter group of receptor proteinsinclude: leukocyte-common antigen (PTPRC; MIM 151460) and leukocyteantigen related tyrosine phosphatase (PTPRF; MIM 179590).

[0037] Protein tyrosine phosphatase zeta (PTPζ) [also known as PTPRZ,HPTP-ZETA, HPTPZ, RPTP-BETA(β), or RPTPB] was isolated as a cDNAsequence by two groups in the early nineties. The complete cDNA sequenceof the protein is provided in SEQ ID NO. 1, and the complete deducedamino acid sequence is provided in SEQ ID NO. 2. Splicing variants andfeatures are indicated in the sequences. Levy et al. (“The cloning of areceptor-type protein tyrosine phosphatase expressed in the centralnervous system” J. Biol. Chem. 268: 10573-10581, (1993)) isolated cDNAclones from a human infant brain step mRNA expression library, anddeduced the complete amino acid sequence of a large receptor-typeprotein tyrosine phosphatase containing 2,307 amino acids.

[0038] Levy found that the protein, which they designated PTP-β (PTPζ),is a transmembrane protein with 2 cytoplasmic PTPase domains and a1,616-amino acid extracellular domain. As in PTP-γ (MIM 176886), the 266N-terminal residues of the extracellular domain are have a high degreeof similarity to carbonic anhydrases (see MIM 114880). The human geneencoding PTPζ has been mapped to chromosome 7q31.3-q32 by chromosomal insitu hybridization (Ariyama et al., “Assignment of the human proteintyrosine phosphatase, receptor-type, zeta (PTPRZ) gene to chromosomeband 7q31.3” Cytogenet. Cell Genet. 70: 52-54 (1995)). Northern blotanalysis has shown that showed that PTP-zeta is expressed only in thehuman central nervous system. By in situ hybridization, Levy et al.(1993) localized the expression to different regions of the adult humanbrain, including the Purkinje cell layer of the cerebellum, the dentategyrus, and the subependymal layer of the anterior horn of the lateralventricle. Levy stated that this was the first mammalian tyrosinephosphatase whose expression is restricted to the nervous system. Inaddition, high levels of expression in the murine embryonic brainsuggest an important role in CNS development.

[0039] Northern analysis has shown three splice variants: theextracellular proteoglycan phosphacan, which contains the fullextracellular region of the protein, and the long (α) and short (β)forms of the transmembrane phosphatase. The β form lacks theextracellular 860 aa long insert domain of the protein, therefore it isnot glycosylated. PCR studies of the gene in rat genomic DNA indicatedthat there are no introns at the putative 5′ and 3′ splice sites or inthe 2.6 kb segment which is deleted in the short transmembrane protein.The phosphatases and the extracellular proteoglycan have different3′-untranslated regions. Additional alternative mRNA splicing is likelyto result in the deletion of a 7 amino acid insert from theintracellular juxtamembrane region of both long and short phosphataseisoforms. Simultaneous quantitation of the three major isoformsindicated that the mRNA encoding phosphacan had the highest relativeabundance in the CNS while that encoding the short phosphatase isoformwas most abundant relative to the other PTPζ variants in the PNS.

[0040] PTPζ has only been found to be expressed in the nervous system.By in situ hybridization, it has been localized to different regions ofthe adult brain, including the Purkinje cell layer of the cerebellum,the dentate gyrus, and the subependymal layer of the anterior horn ofthe lateral ventricle. High levels of PTPζ have been seen in regions ofthe brain where there is continued neurogenesis and neurite outgrowth,and it seems to play a role in morphogenesis and plasticity of thenervous system. Phosphacan immunoreactivity has been associated withperineuronal nets around parvalbumin-expressing neurons in adult ratcerebral cortex. Neurons as well as astrocytes have been shown toexpress phosphacan.

[0041] The transmembrane forms of PTPζ are expressed on the migratingneurons especially at the lamellipodia along the leading processes. PTPζis postulated to be involved in the neuronal migration as a neuronalreceptor of pleiotrophin distributed along radial glial fibers. PTPζ hasbeen shown to be highly expressed in radial glia and other forms ofglial cells that play an important role during development. Theanti-PTPζ staining localizes to the radial processes of these cells,which act as guides during neuronal migration and axonal elongation. Thepattern of RPTP-zeta expression has also been shown to change with theprogression of glial cell differentiation.

[0042] The three splicing variants of RPTP-zeta have been shown to havedifferent spatial and temporal patterns of expression in the developingbrain. The 9.5-kb and 6.4-kb transcripts, which encode the α and βtransmembrane protein tyrosine phosphatases, were predominantlyexpressed in glial progenitors located in the subventricular zone. The8.4-kb transcript, which encodes the secreted chondroitin sulfateproteoglycan phosphacan, was expressed at high levels by more matureglia that have migrated out of the subventricular zone. The threetranscripts have also been shown to be differentially expressed in glialcell cultures.

[0043] In knockout studies, PTPζ-deficient mice were viable, fertile,and showed no gross anatomical alterations in the nervous system orother organs. Therefore, it was deduced that PTPζ is not essential forneurite outgrowth and node formation in mice. The ultrastructure ofnerves of the central nervous system in PTPζ-deficient mice suggests afragility of myelin. However, conduction velocity was not altered. Thenormal development of neurons and glia in was thought to indicate thatPTPζ function is not necessary for these processes in vivo, or that aloss of PTPζ can be compensated for by other protein tyrosinephosphatases expressed in the nervous system.

[0044] Following CNS injury, robust induction of phosphatase forms ofPTPζ mRNA has been observed in areas of axonal sprouting, and of bothphosphatases and phosphacan mRNAs in areas of glial scarring. This isthought to imply that the encoded proteins and the cell adhesionmolecules and extracellular matrix proteins to which they bind maycontribute to recovery from injury and perhaps also to the regulation ofaxonal regrowth in the nervous system. Following peripheral nerve crush,all PTPζ mRNAs, including phosphacan and the phosphatase variants withand without the 21 base insert, were observed to be significantlyinduced in the distal segments of the sciatic nerve with a time coursethat correlated well with the response of Schwann cells to this injury.

[0045] The extracellular domains of PTPζ have been shown to be capableof binding to several cell adhesion molecules. Phosphacan, which is theshortest, secreted form of PTPζ, containing the full extracellularregion, previously was designated 3F8 and 6B4 chondroitin sulfateproteoglycan or 3H1 keratin sulfate proteoglycan depending on theglycosylation status. It is synthesized mainly by glia and binds toneurons and to the neural cell adhesion molecules Ng-CAM/L1, NCAM,TAG-1/axonin-1, to tenascin-C and R, to amphoterin andpleiotrophin/heparin-binding growth-associated molecule (HB-GAM)(amphoterin and pleiotrophin are heparin-binding proteins that aredevelopmentally regulated in brain and functionally involved in neuriteoutgrowth). Binding of phosphacan to Ng-CAM/L1, NCAM, and tenascin-C(FNIII domain) is mediated by complex-type N-linked oligosaccharides onthe proteoglycan. Phosphacan, shows saturable, reversible, high-affinitybinding to fibroblast growth factor-2 (FGF-2). The interaction ismediated primarily through the core protein. Immunocytochemical studieshave also shown an overlapping localization of FGF-2 and phosphacan inthe developing central nervous system. The core protein of phosphacanmay also regulate the access of FGF-2 to cell surface signalingreceptors in nervous tissue.

[0046] The carbonic anhydrase (CAH) domain of PTPζ has been shown tobind specifically to contactin. Contactin is a 140 kDa GPImembrane-anchored neuronal cell recognition protein expressed on thesurface of neuronal cells. The CAH domain of RPTP zeta was shown toinduce cell adhesion and neurite growth of primary tectal neurons, anddifferentiation of neuroblastoma cells. These responses were blocked byantibodies against contactin, demonstrating that contactin is a neuronalreceptor for RPTP zeta. Caspr ((p190/Caspr, a contactin-associatedtransmembrane receptor) and contactin exist as a complex in rat brainand are bound to each other by means of lateral (cis) interactions inthe plasma membrane. The extracellular domain of Caspr contains aneurophilin/coagulation factor homology domain, a region related tofibrinogen beta/gamma, epidermal growth factor-like repeats, neurexinmotifs as well as unique PGY repeats found in a molluscan adhesiveprotein. The cytoplasmic domain of Caspr contains a proline-richsequence capable of binding to a subclass of SH3 domains of signalingmolecules. Caspr may function as a signaling component of contactin,enabling recruitment and activation of intracellular signaling pathwaysin neurons. The role of the extracellular domains in neural adhesion andneurite growth induction was investigated by the use of fusion proteinconstructs. The results suggested that binding of glial PTPζ to thecontactin/Nr-CAM complex is important for neurite growth and neuronaldifferentiation.

[0047] PTPζ was shown to bind to a heparin-binding growth factor midkinethrough the chondroitin sulfate portion of the receptor. Theinteractions of pleiotrophin (PTN) with the receptor in U373-MG cellswas also studied. Pleiotrophin was shown to bind to the spacer domain.Results suggested that PTN signals through “ligand-dependent receptorinactivation” of PTPζ and disrupts its normal roles in the regulation ofsteady-state tyrosine phosphorylation of downstream signaling molecules.PTN was shown to bind to and functionally inactivate the catalyticactivity of PTPζ. An active site-containing domain of PTPζ both bindsβ-catenin and functionally reduces its levels of tyrosinephosphorylation when added to lysates of pervanadate-treated cells. Inunstimulated cells, PTPζ was shown to be intrinsically active, andthought to function as an important regulator in the reciprocal controlof the steady-state tyrosine phosphorylation levels of β-catenin bytyrosine kinases and phosphatases.

[0048] Using the yeast substrate-trapping system, several substratecandidates for PTPζ were isolated. The results indicated that GIT1/Cat-1is a substrate molecule of PTPζ. In addition, PTPζ was shown to bind tothe PSD-95/SAP90 family through the second phosphatase domain.Immunohistochemical analysis revealed that PTPζ and PSD-95/SAP90 aresimilarly distributed in the dendrites of pyramidal neurons of thehippocampus and neocortex. Subcellular fractionation experimentsindicated that PTPζ is concentrated in the postsynaptic densityfraction. These results suggested that PTPζ is involved in theregulation of synaptic function as postsynaptic macromolecular complexeswith PSD-95/SAP90.

[0049] Voltage-gated sodium channels in brain neurons were also found toassociate with the membrane bound forms of PTPζ and phosphacan. Both theextracellular domain and the intracellular catalytic domain of PTPζinteracted with sodium channels. Sodium channels were tyrosinephosphorylated and were modulated by the associated catalytic domains ofPTPζ.

SUMMARY OF THE INVENTION

[0050] The present invention provides novel methods and reagents forspecifically targeting brain tumor neoplastic cells for both therapeuticand imaging purposes, by targeting brain tumor protein targets (T_(BT)).These targets have been identified by the applicants as beingoverexpressed in brain tumors, and thus allow for the selectiveinhibition of cell function or selective marking for visualization withtherapeutic or visualizing compositions which have a specific affinityfor thes eprotein targets. Each of angiopoietin related protein 2(ARP-2,) secreted protein acidic, rich in cysteine (SPARC,) c-metproto-oncogene (C-MET,) brevican (BEHAB,) CD-44 antigen (CD-44,)tetraspanin 3 (TSPN3,) pleiotrophin (PTN,) osteopontin (OPN,) vasoactiveintestinal peptide receptor-2 (VIPR-2,) and receptor protein tyrosinephosphatase zeta (PTPζ) [including the two novel isoforms PTPζ SM1 andSM2], as the proteins are described below, have been identified as anindependently useful protein target T_(BT). In some preferredembodiments of the invention, either novel isofom PTPζ SM1 or PTPζ SM2is the protein target T_(BT). Thus, the aspects of the invention withrelation to each of these T_(BT) are described generally as follows:

[0051] In a first aspect, the present invention provides T_(BT)affinity-based compounds and compositions useful in treating a braintumor in a patient. The compositions and compounds of this aspect of theinvention generally fall into two groups: T_(BT)-binding conjugatecompounds, which comprise a cytotoxic moiety (C), which inhibits thegrowth of tumor cells; and T_(BT)-binding compound compositions in whichthe T_(BT) binding moiety alters the normal function of the T_(BT) in oraround the tumor cell, thus inhibiting cell growth and/or function.

[0052] In a first group of embodiments of this aspect of the invention,T_(BT)-binding therapeutic conjugate compounds are provided. Thesecompounds have the general formula α(T_(BT))C, wherein α(T_(BT)) is oneor more moieties which specifically binds to a T_(BT), and C is one ormore cytotoxic moieties. In preferred embodiments, α(T_(BT)) is anantibody or an antibody fragment. In particularly preferred embodiments,α(T_(BT)) is an antibody or an antibody fragment which elicits a reducedimmune response when administered to a human patient. Preferredcytotoxic moieties for use in these embodiments of the invention includeradioactive moieties, chemotoxic moieties, and toxin proteins. Theinvention also provides compositions comprising these T_(BT)-bindingtherapeutic conjugate compounds in a pharmaceutically acceptablecarrier.

[0053] In a second group of embodiments of this first aspect of theinvention, T_(BT)-binding therapeutic compounds are provided which alterthe normal function of the T_(BT) in or around brain tumor cells andinhibit brain tumor cell growth. These T_(BT) -binding therapeuticcompounds have the general formula α(T_(BT)), wherein α(T_(BT)) is oneor more moieties which specifically binds to a T_(BT), and wherein thebinding of α(T_(BT)) alters the function of the T_(BT). In preferredembodiments, α(T_(BT)) is an antibody or an antibody fragment. Inparticularly preferred embodiments, α(T_(BT)) is an antibody or anantibody fragment which elicits a reduced immune response whenadministered to a human patient. It is preferred that the therapeuticcompounds of this second group of embodiments of the first aspect of theinvention be formulated into therapeutic compositions comprising theT_(BT)-binding compound in a pharmaceutically acceptable carrier.

[0054] In a second aspect, the present invention provides methods forusing these compounds and compositions to treat a brain tumor in apatient. The methods comprise administering an effective amount of acomposition, comprising a T_(BT)-binding compound from the first orsecond group of embodiments of the first aspect and a pharmaceuticallyacceptable carrier, to a patient in need thereof. Brain tumors treatedin this fashion may be glioblastomas, astrocytomas, neuroblastomas, orany type of brain tumor. Administration of the therapeutic compositionmay be by any acceptable means. One preferred method for administrationis by intrathecal administration, although intravascular administrationis also preferred.

[0055] In a third aspect, the present invention provides T_(BT)affinity-based compounds and compositions for the visualization of braintumors in patients. These compounds have the general formula α(T_(BT))I,wherein α(T_(BT)) is one or more moieties which specifically binds to aT_(BT), and I is one or more imaging moieties. In preferred embodiments,α(T_(BT)) is an antibody or an antibody fragment. In particularlypreferred embodiments, αA(T_(BT)) is an antibody or an antibody fragmentwhich elicits a reduced immune response when administered to a humanpatient. Preferred I moieties include radiographic moieties (useful in,e.g., x-ray, scintillation, or other radiation imaging methods,)positron-emitting moieties, magnetic spin contrast moieties, andoptically visible moieties (such as visible particles, fluorescent dyes,and visible-spectrum dyes.) It is preferred that the imaging compoundsof these embodiments of the third aspect of the invention be formulatedinto therapeutic compositions comprising the T_(BT)-binding compound ina pharmaceutically acceptable carrier.

[0056] In a fourth aspect, the present invention provides methods ofusing the compounds and compositions of the third aspect of theinvention to visualize a brain tumor in a patient. These methodsgenerally comprise administering an effective amount of an imagingcompound of the general formula α(T_(BT))I in a pharmaceuticallyacceptable carrier to the patient, and then visualizing the imagingmoieties of the compound. Administration of the imaging composition maybe by any acceptable means. Intravascular administration of the imagingcomposition is preferred in these methods, although intrathecaladministration is also preferred. Preferred methods of visualizing theimaging moieties of the compounds include radiographic imagingtechniques (e.g., x-ray imaging and scintillation imaging techniques),positron-emission tomography, magnetic resonance imaging techniques, anddirect or indirect (e.g., endoscopic) visual inspection.

[0057] Various particular embodiments of these aspects of the inventioninclude:

[0058] A method to treat a brain tumor by administering a therapeuticamount of a composition comprising a compound of the general formulaα(ARP2)C, wherein α(ARP2) is one or more moieties which specificallybinds to a human angiopoietin related protein-2, and C is one or morecytotoxic moieties, and a pharmaceutically acceptable carrier.

[0059] A compound for the treatment of a brain tumor of the generalformula α(ARP2)C, wherein α(ARP2) is one or more moieties whichspecifically binds to a human angiopoietin related protein-2, and C isone or more cytotoxic moieties.

[0060] A method to treat a brain tumor by administering a therapeuticamount of a composition comprising a compound of the general formulaα(ARP2), wherein α(ARP2) is one or more moieties which specificallybinds to a human angiopoietin related protein-2, wherein the binding ofα(ARP2) alters the function of the angiopoietin related protein-2, and apharmaceutically acceptable carrier.

[0061] A method for visualizing a brain tumor in a patient by firstadministering to a patient an effective amount of a compositioncomprising: a compound of the general formula α(ARP2)I, wherein α(ARP2)is one or more moieties which specifically binds to a human angiopoietinrelated protein-2, and I is one or more imaging moieties and apharmaceutically acceptable carrier, and then visualizing the imagingmoieties of the compound.

[0062] A composition for the visualization of a brain tumor comprising acompound of the general formula α(ARP2)I, wherein α(ARP2) is one or moremoieties which specifically binds to a human angiopoietin relatedprotein-2, and I is one or more imaging moieties, and a pharmaceuticallyacceptable carrier.

[0063] A method to treat a brain tumor by administering a therapeuticamount of a composition comprising a compound of the general formulaα(TSPAN3)C, wherein α(TSPAN3) is one or more moieties which specificallybinds to a human tetraspanin 3, and C is one or more cytotoxic moieties,and a pharmaceutically acceptable carrier.

[0064] A compound for the treatment of a brain tumor of the generalformula α(TSPAN3)C, wherein α(TSPAN3) is one or more moieties whichspecifically binds to a human tetraspanin 3, and C is one or morecytotoxic moieties.

[0065] A method to treat a brain tumor by administering a therapeuticamount of a composition comprising a compound of the general formulaα(TSPAN3), wherein α(TSPAN3) is one or more moieties which specificallybinds to a human tetraspanin 3, wherein the binding of α(TSPAN3) altersthe function of the tetraspanin 3, and a pharmaceutically acceptablecarrier.

[0066] A composition for the treatment of a brain tumor comprising: acompound of the general formula α(TSPAN3), wherein α(TSPAN3) is one ormore moieties which specifically binds to a human tetraspanin 3, whereinthe binding of α(TSPAN3) alters the function of the tetraspanin 3, and apharmaceutically acceptable carrier.

[0067] A method for visualizing a brain tumor in a patient by firstadministering to a patient an effective amount of a compositioncomprising: a compound of the general formula α(TSPAN3)I, whereinα(TSPAN3) is one or more moieties which specifically binds to a humantetraspanin 3, and I is one or more imaging moieties, and apharmaceutically acceptable carrier, and then visualizing the imagingmoieties of the compound.

[0068] A composition for the visualization of a brain tumor comprising acompound of the general formula α(TSPAN3)I, wherein α(TSPAN3) is one ormore moieties which specifically binds to a human tetraspanin 3, and Iis one or more imaging moieties, and a pharmaceutically acceptablecarrier.

[0069] A method to treat a brain tumor by administering a therapeuticamount of a composition comprising a compound of the general formulaα(PTPζ)C, wherein α(PTPζ) is one or more moieties which specificallybinds to a human PTPζ selected from the group consisting of PTPζ SM1 andPTPζ SM2, further wherein α(PTPζ) does not specifically bind to humanPTPζ α, human PTPζ β, or phosphacan, and C is one or more cytotoxicmoieties, and a pharmaceutically acceptable carrier.

[0070] A compound for the treatment of a brain tumor of the generalformula α(PTPζ)C, wherein α(PTPζ) is one or more moieties whichspecifically binds to a human PTPζ selected from the group consisting ofPTPζ SM1 And PTPζ SM2, further wherein α(PTPζ) does not specificallybind to human PTPζ α, human PTPζ β,or human phosphacan, and C is one ormore cytotoxic moieties.

[0071] A method to treat a brain tumor by administering a therapeuticamount of a composition comprising a compound of the general formulaα(PTPζ), wherein α(PTPζ) is one or more moieties which specificallybinds to a human PTPζ selected from the group consisting of PTPζ SM1 AndPTPζ SM2, further wherein α(PTPζ) does not specifically bind to humanPTPζ α, human PTPζ, or phosphacan, wherein the binding of α(PTPζ) altersthe function of the human PTPζ,and a pharmaceutically acceptablecarrier.

[0072] A composition for the treatment of a brain tumor comprising acompound of the general formula α(PTPζ), wherein α(PTPζ) is one or moremoieties which specifically binds to PTPζ selected from the groupconsisting of PTPζ SM1 And PTPζ SM2, further wherein α(PTPζ) does notspecifically bind to human PTPζ α, human PTPζ α, or phosphacan, whereinthe binding of α(PTPζ) alters the function of the PTPζ, and apharmaceutically acceptable carrier.

[0073] A method for visualizing a brain tumor in a patient by firstadministering to a patient an effective amount of a compositioncomprising: a compound of the general formula α(PTPζ)I, wherein α(PTPζ)is one or more moieties which specifically binds to a human PTPζselected from the group consisting of PTPζ SM1 And PTPζ SM2, furtherwherein α(PTPζ) does not specifically bind to human PTPζ α, human PTPζβ, or phosphacan, and I is one or more imaging moieties and apharmaceutically acceptable carrier, and then visualizing the imagingmoieties of the compound.

[0074] A composition for the visualization of a brain tumor comprising acompound of the general formula α(PTPζ)I, wherein α(PTPζ) is one or moremoieties which specifically binds to a human PTPζ selected from thegroup consisting of PTPζ SM1 And PTPζ SM2, further wherein α(PTPζ) doesnot specifically bind to human PTPζ α, human PTPζ β, or phosphacan, andI is one or more imaging moieties, and a pharmaceutically acceptablecarrier.

[0075] A method to treat a brain tumor by administering a therapeuticamount of a composition comprising a compound of the general formulaα(SPARC)C, wherein α(SPARC) is one or more moieties which specificallybinds to a human secreted protein, rich in cysteine, and C is one ormore cytotoxic moieties, and a pharmaceutically acceptable carrier.

[0076] A compound for the treatment of a brain tumor of the generalformula α(SPARC)C, wherein α(SPARC) is one or more moieties whichspecifically binds to a human secreted protein, rich in cysteine, and Cis one or more cytotoxic moieties.

[0077] A method to treat a brain tumor by administering a therapeuticamount of a composition comprising a compound of the general formulaα(SPARC), wherein α(SPARC) is one or more moieties which specificallybinds to a human secreted protein, rich in cysteine, wherein the bindingof α(SPARC) alters the function of the secreted protein, rich incysteine, and a pharmaceutically acceptable carrier.

[0078] A method for visualizing a brain tumor in a patient by firstadministering to a patient an effective amount of a compositioncomprising: a compound of the general formula α(SPARC)I, whereinα(SPARC) is one or more moieties which specifically binds to a humansecreted protein, rich in cysteine, and I is one or more imagingmoieties, and a pharmaceutically acceptable carrier, and thenvisualizing the imaging moieties of the compound.

[0079] A composition for the visualization of a brain tumor comprising acompound of the general formula α(SPARC)I, wherein α(SPARC) is one ormore moieties which specifically binds to a human secreted protein, richin cysteine, and I is one or more imaging moieties, and apharmaceutically acceptable carrier.

[0080] A method to treat a brain tumor by administering a therapeuticamount of a composition comprising a compound of the general formulaα(c-MET)C, wherein α(c-MET) is one or more moieties which specificallybinds to a human c-MET oncogene product, and C is one or more cytotoxicmoieties and a pharmaceutically acceptable carrier.

[0081] A method to treat a brain tumor by administering a therapeuticamount of a composition comprising a compound of the general formulaα(c-MET), wherein α(c-MET) is one or more moieties which specificallybinds to a human c-MET oncogene product, wherein the binding ofα(TSPAN3) alters the function of the c-MET oncogene product, and apharmaceutically acceptable carrier.

[0082] A method for visualizing a brain tumor in a patient by firstadministering to a patient an effective amount of a compositioncomprising: a compound of the general formula α(c-MET)I, whereinα(c-MET) is one or more moieties which specifically binds to a humanc-MET oncogene product, and I is one or more imaging moieties and apharmaceutically acceptable carrier, and then visualizing the imagingmoieties of the compound.

[0083] A method to treat a brain tumor by administering a therapeuticamount of a composition comprising a compound of the general formulaα(CD44)C, wherein α(CD44) is one or more moieties which specificallybinds to a human CD44 antigen, and C is one or more cytotoxic moieties,and a pharmaceutically acceptable carrier.

[0084] A method to treat a brain tumor by administering a therapeuticamount of a composition comprising a compound of the general formulaα(CD44), wherein α(CD44) is one or more moieties which specificallybinds to a human CD44 antigen, wherein the binding of α(CD44) alters thefunction of the CD44 antigen, and a pharmaceutically acceptable carrier.

[0085] A method for visualizing a brain tumor in a patient by firstadministering to a patient an effective amount of a compositioncomprising: a compound of the general formula α(CD44)I, wherein α(CD44)is one or more moieties which specifically binds to a human CD44antigen, and I is one or more imaging moieties, and a pharmaceuticallyacceptable carrier, and then visualizing the imaging moieties of thecompound.

[0086] A method to treat a brain tumor by administering a therapeuticamount of a composition comprising a compound of the general formulaα(VIPR2)C, wherein α(VIPR2) is one or more moieties which specificallybinds to a human vasoactive intestinal peptide receptor-2, and C is oneor more cytotoxic moieties and a pharmaceutically acceptable carrier.

[0087] A method to treat a brain tumor by administering a therapeuticamount of a composition comprising a compound of the general formulaα(VIPR2), wherein α(VIPR2) is one or more moieties which specificallybinds to a human vasoactive intestinal peptide receptor-2, wherein thebinding of α(VIPR2) alters the function of the vasoactive intestinalpeptide receptor-2, and a pharmaceutically acceptable carrier.

[0088] A method for visualizing a brain tumor in a patient by firstadministering to a patient an effective amount of a compositioncomprising: a compound of the general formula α(VIPR2)I, whereinα(VIPR2) is one or more moieties which specifically binds to a humantetraspanin 3, and I is one or more imaging moieties, and apharmaceutically acceptable carrier, and then visualizing the imagingmoieties of the compound.

[0089] A method to treat a brain tumor by administering a therapeuticamount of a composition comprising a compound of the general formulaα(OPN)C, wherein α(OPN) is one or more moieties which specifically bindsto a human osteopontin, and C is one or more cytotoxic moieties and apharmaceutically acceptable carrier.

[0090] A compound for the treatment of a brain tumor of the generalformula α(OPN)C, wherein (OPN) is one or more moieties whichspecifically binds to a human tetraspanin 3, and C is one or morecytotoxic moieties.

[0091] A method to treat a brain tumor by administering a therapeuticamount of a composition comprising a compound of the general formulaα(OPN), wherein α(OPN) is one or more moieties which specifically bindsto a human osteopontin, wherein the binding of α(OPN) alters thefunction of the osteopontin, and a pharmaceutically acceptable carrier.

[0092] A method for visualizing a brain tumor in a patient by firstadministering to a patient an effective amount of a compositioncomprising: a compound of the general formula α(OPN)I, wherein α(OPN) isone or more moieties which specifically binds to a human osteopontin,and I is one or more imaging moieties, and a pharmaceutically acceptablecarrier, and then visualizing the imaging moieties of the compound.

[0093] A composition for the visualization of a brain tumor comprising acompound of the general formula α(OPN)I, wherein α(OPN) is one or moremoieties which specifically binds to a human osteopontin, and I is oneor more imaging moieties, and a pharmaceutically acceptable carrier.

[0094] A method to treat a brain tumor by administering a therapeuticamount of a composition comprising a compound of the general formulaα(PTN)C, wherein α(PTN) is one or more moieties which specifically bindsto a human pleiotrophin, and C is one or more cytotoxic moieties, and apharmaceutically acceptable carrier.

[0095] A compound for the treatment of a brain tumor of the generalformula α(PTN)C, wherein α(PTN) is one or more moieties whichspecifically binds to a human pleiotrophin, and C is one or morecytotoxic moieties.

[0096] A method to treat a brain tumor by administering a therapeuticamount of a composition comprising a compound of the general formulaα(PTN), wherein α(PTN) is one or more moieties which specifically bindsto a human pleiotrophin, wherein the binding of α(PTN) alters thefunction of the pleiotrophin, and a pharmaceutically acceptable carrier.

[0097] A method for visualizing a brain tumor in a patient by firstadministering to a patient an effective amount of a compositioncomprising: a compound of the general formula α(PTN)I, wherein α(PTN) isone or more moieties which specifically binds to a human pleiotrophin,and I is one or more imaging moieties, and a pharmaceutically acceptablecarrier, and then visualizing the imaging moieties of the compound.

[0098] A composition for the visualization of a brain tumor comprising acompound of the general formula α(PTN)I, wherein α(PTN) is one or moremoieties which specifically binds to a human pleiotrophin, and I is oneor more imaging moieties, and a pharmaceutically acceptable carrier.

[0099] Brain tumors are known to be relatively heterogeneous, and thusall patients may not respond the same to a particular protein targettreatment. Thus, in addition to the independent uses of each of theT_(BT) protein targets as described above, the invention also provides,in yet another aspect, combination therapeutic and/or visualizationagents, compositions, and methods. These combination embodiments of theinvention may utilize as brain tumor protein targets any two or more ofthe identified targets angiopoietin related protein 2 (ARP-2,) secretedprotein acidic, rich in cysteine (SPARC,) c-met proto-oncogene (c-MET,)brevican (BEHAB,) cd-44 antigen (CD-44,) tetraspanin 3 (TSPN3,)pleiotrophin (PTN,) osteopontin (OPN,) vasoactive intestinal peptidereceptor-2 (VIPR-2,) and receptor protein tyrosine phosphatase zeta(PTPζ) [including the two novel isoforms PTPζ SM1 and SM2]. In somepreferred embodiments, the brain tumor protein targets are selected fromthe group consisting of angiopoietin related protein 2 (ARP-2,) c-metproto-oncogene (c-MET,) cd-44 antigen (CD-44,) tetraspanin 3 (TSPN3,)osteopontin (OPN,) and receptor protein tyrosine phosphatase zeta(PTPζ). Embodiments of the combination aspect of the invention maytarget a group of proteins from the identified targets with similarcompartmentalization characteristics. Thus, the combination aspects maytarget two or more secreted proteins from the group angiopoietin relatedprotein 2 (ARP-2,) secreted protein acidic, rich in cysteine (SPARC,)brevican (BEHAB,) pleiotrophin (PTN,) secreted forms of receptor proteintyrosine phosphatase zeta (PTPζ) [including the novel isoform PTPζ SM1].Or, the combination aspects may target one or more of the extracellularmatrix binding proteins secreted protein acidic, rich in cysteine(SPARC,) and/or brevican (BEHAB,) with one or more of the otheridentified protein targets. Or, the combination aspects may target oneor more of the membrane-bound proteins from the group c-metproto-oncogene (c-MET,) cd-44 antigen (CD-44,) tetraspanin 3 (TSPN3,)osteopontin (OPN,) vasoactive intestinal peptide receptor-2 (VIPR-2,)and membrane bound forms of receptor protein tyrosine phosphatase zeta(PTPζ) [including the novel isoform PTPζ SM2].

[0100] In preferred embodiments, at least one of the protein targets isselected from the proteins angiopoietin related protein 2 (ARP-2,)tetraspanin 3 (TSPN3,), and receptor protein tyrosine phosphatase zeta(PTPζ) [including the two novel isoforms PTPζ SM1 and SM2]. In otherpreferred embodiments, one of these proteins and another proteinselected from the secreted group, the extracellular matrix group, or themembrane-bound group. In another group of preferred embodiments arecombination aspects targeting two or more of angiopoietin relatedprotein 2 (ARP-2,) tetraspanin 3 (TSPN3,), and receptor protein tyrosinephosphatase zeta (PTPζ) [including the two novel isoforms PTPζ SM1 andSM2]. Another group of preferred embodiments are combination aspectswhich target angiopoietin related protein 2 (ARP-2), and one or moreproteins selected from the group secreted protein acidic, rich incysteine (SPARC,) c-met proto-oncogene (C-MET,) brevican (BEHAB,) CD-44antigen (CD-44,) tetraspanin 3 (TSPN3,) pleiotrophin (PTN,) osteopontin(OPN,) vasoactive intestinal peptide receptor-2 (VIPR-2,) and receptorprotein tyrosine phosphatase zeta (PTPζ) [including the two novelisoforms PTPζ SM1 and SM2]. Another group of preferred embodiments arecombination aspects which target tetraspanin 3 (TSPN3), and one or moreproteins selected from the group secreted protein acidic, rich incysteine (SPARC,) c-met proto-oncogene (C-MET,) brevican (BEHAB,) CD-44antigen (CD-44,) angiopoietin related protein 2 (ARP-2,) pleiotrophin(PTN,) osteopontin (OPN,) vasoactive intestinal peptide receptor-2(VIPR-2,) and receptor protein tyrosine phosphatase zeta (PTPζ)[including the two novel isoforms PTPζ SM1 and SM2]. Another group ofpreferred embodiments are combination aspects which target receptorprotein tyrosine phosphatase zeta (PTPζ) [including the two novelisoforms PTPζ SM1 and SM2] and one or more proteins selected from thegroup angiopoietin related protein 2 (ARP-2,) secreted protein acidic,rich in cysteine (SPARC,) c-met proto-oncogene (C-MET,) brevican(BEHAB,) CD-44 antigen (CD-44,) tetraspanin 3 (TSPN3,) pleiotrophin(PTN,) osteopontin (OPN,) and vasoactive intestinal peptide receptor-2(VIPR-2.) As pleiotrophin (PTN) is a known ligand of PTPζ, anotherpreferred embodiment of the combination aspects of the inventionutilizes these proteins as targets, either alone or in combination withone or more of the other identified targets.

[0101] In yet another aspect, the present invention provides two novelsplicing isoforms of PTPζ, shown to be expressed in brain tissue. Thesenovel isoforms, PTPζ SM1 and PTPζ SM2, described in more detail below,differ in structure from the three known isoforms heretofore disclosed.PTPζ SM1 comprises the amino acids encoded by the first nine exons ofPTPζ-α, with three unique additional carboxy terminal amino acidsencoded by additional 3′ mRNA sequence from the intron of the genebetween exons nine and ten. The mRNA for PTPζ SM2 comprises all exons ofPTPζ-α, with a 116 nucleotide insertion, in the correct reading frame,in the mRNA sequence between exons 23 and 24, from the from the intronof the gene between exons 23 and 24. Thus, embodiments of this aspect ofthe invention include the mature proteins of PTPζ splice variants SM1 orSM2, and nucleic acids encoding these novel spice variants, as wellproteins with significant homology to the splice variants.

[0102] Thus, in one group of embodiments of this aspect, the inventionprovides nucleic acid polymers comprising the sequence of nucleotides148 to 1272 of SEQ ID NO. 1, the complement of nucleotides 148 to 1272of SEQ ID NO. 1, nucleotides 148 to 7209 of SEQ ID NO. 3, or thecomplement of nucleotides 148 to 7209 of SEQ ID NO. 3. In another groupof embodiments of this aspect, the invention provides polypeptidescomprising the amino acid sequence of SEQ ID NO. 2 or the amino acidsequence of SEQ ID NO. 4.

[0103] In an additional related aspect, the invention providespolypeptides comprising a distinctive portion of the amino acid sequenceof SEQ ID NO. 2 or SEQ ID NO. 4. Such peptides are useful for theproduction of antibodies against the PTPζ SM1 or SM2 splicing variants.Preferably, these polypeptides comprise a portion of the amino acidsequence of SEQ ID NO. 2 or SEQ ID NO. 4 which is at least 6, morepreferably at least 8, more preferably at least 10, more preferably atleast 15, and most preferably at lest 20 amino acids in length. In somepreferred embodiments of this aspect of the invention, the polypeptidescomprise the three unique terminal amino acids of PTPζ SM1 after exon 9.In other preferred embodiments, the polypeptides comprise a portion ofthe unique exon 23a of PTPζ SM2, wherein the portion is preferably atleast 3 amino acids in length, more preferably at least 6 amino acids inlength, more preferably at least 9 amino acids in length, and mostpreferably at least 15 amino acids in length.

[0104] In an additional related aspect, the invention also providesaffinity reagents which specifically bind to PTPζ splice variants SM1 orSM2, but do not bind to the other known splice variants of PTPζ (e.g.,α, β, or phosphacan forms). In preferred embodiments these affinityreagents are antibodies or antibody fragments.

[0105] In an additional related aspect, the invention also providesnucleic acid sequences encoding the PTPζ splice variants SM1 or SM2. Theinvention also encompasses nucleic acid probes which hybridize to themRNA encoding PTPζ splice variants SM1 or SM2, but not mRNA encodingother known splice variants of PTPζ.

BRIEF DESCRIPTION OF THE FIGURES

[0106]FIG. 1: A diagram of the three known splicing variant isoforms ofPTPζ. The approximate position of the domains of the isoforms isindicated underneath the isoforms, as well as the approximate exon size(for size reference, exon 12 is 3.6 kilobases.) Isoform PTPζ-α is thefull length isoform, which contains the primary amino acid sequence aa25-2314 of SEQ ID NO. 2 (aa 1-24 are a signal polypeptide). In IsoformPTPζ-β, aa 755-1614 are missing. Isoform PTPζ-S (phosphacan), is asecreted isoform which comprise the extracellular domains of PTPζ-α, inwhich the transmembrane and cytosol domains are missing.

[0107]FIG. 2: A diagram of the two newly discovered splicing variantisoforms of PTPζ. The approximate position of the domains of theisoforms is indicated underneath the isoforms, as well as theapproximate exon size (for size reference, exon 12 is 3.6 kilobases.) SM1 fails to splice correctly after the 9^(th) exon, yielding an mRNA withtow extra codons followed by a stop codon after the normal terminus ofexon 9. SM 2 contains a 116 nucleotide insertion from between exons 23&24.

[0108]FIG. 3: A diagram comparing the three known PTPζ isoforms with thetwo novel isoforms.

DETAILED DESCRIPTION OF THE INVENTION

[0109] Applicants have identified several brain tumor protein targetsand genes which are differentially regulated between brain cancer tissue(glioblastoma) and normal brain tissue. Applicants have performeddifferential cloning between cancerous and normal brains and haveidentified the brain tumor protein target genes by DNA sequenceanalysis. Based on the observation in other diseases, particularly othercancers, in which overexpressed genes can contribute to the pathology ofthe disease, these overexpressed genes and their protein productsmediate the initiation and progression of brain tumors. Thus, theoverexpressed brain tumor protein targets, which are presented on thecell surface, provide excellent targets for immunotherapeutic agentswhich either deliver cytotoxic agents to directly promote tumor celldeath, or which alter the function of the brain tumor protein targets toinhibit the normal physiology of the tumor cell. In addition,immunoimaging agents targeted to the brain tumor protein targets may beutilized to visualize the tumor mass either in diagnostic methods (e.g.,magnetic resonance imaging (MRI) or radiography), or in surgery (e.g.,by the use of optically visual dye moieties in the immunoimaging agent).

[0110] Applicants have identified the brain tumor protein targets by adirect examination of the expression level of genes in actual tumorcells. These samples provide a more accurate and realistic picture oftumor cell biology, especially on the detailed transcriptome level, thananimal models or established cell tissue culture cell lines. Severalgroups have found that cell lines established from astrocytomas andother cell lines do not exhibit expression patterns which reflect theactual expression of the original tumor. For instance, Schreiber, et.al., “Primary brain tumors differ in their expression of octamerdeoxyribonucleic acid-binding transcription factors from long-termcultured glioma cell lines.” Neurosurgery 34: 129-35 (1994), showed thatnervous system-specific transcription factors known as N-Oct proteinsare differentially expressed in human neuroblastoma and glioblastomacell lines in vitro. However, when these results were compared tofreshly isolated human primary and metastatic brain tumors, of the fiveastrocytomas and three glioblastomas analyzed, all but two tumorsdisplayed the complete N-Oct protein profile, irrespective ofhistopathological tumor grade. Similarly, Eberle, et al., “Theexpression of angiogenin in tissue samples of different brain tumors andcultured glioma cells.”Anticancer Res 20: 1679-84 (2000), could showthat angiogenin is detectable in different kinds of intracranial tumortissue samples. Although angiogenin could be detected in primarycultivated glioma cells, it was not detected in the permanent celllines. Finally, Hartmann, et al., “The rate of homozygous CDKN2A/p16deletions in glioma cell lines and in primary tumors.”Int J Oncol 15:975-82 (1999), showed that the rate of homozygous deletions ofCDKN2A/p16 is variable between different tumor entities, but the rate ofdeletions is higher in established cell lines in comparison with primarytumors. Hartmann hypothesized that such incongruity may reflectstatistical sampling errors, true differences depending on tissuederivatization and CDKN2A/p16 loss under selective pressure in tissueculture. After comparing established cell lines derived from humanglioblastomas and their corresponding primary tumors by multiplex PCRmethodology, they found that in 2 of 11 cases (18%) the primary tumorhad no p16 alteration whereas the corresponding cell lines had ahomozygous p16 deletion, and that CDKN2A/p16 was lost already in theearliest passages of the cell lines. Thus, Hartmann concluded that thedeletion was the result of selective cell-culture pressures in manycases.

[0111] These inconsistent results arise because the tumor tissue samplesare obtained from their native milieu, without allowing them theopportunity to alter their gene expression levels in response toartificial environmental stimuli. As recently reported by the BrainTumor Progress Review group of the National Cancer Institute inNovember, 2000, conventionally used glioblastoma cell lines containgenetic and gene expression alterations that are well defined and do notnecessarily reflect the primary tumors from which they were derived. Inaddition, these cell lines are highly homogenous, unlike a primary braintumor. Therefore, data derived soley from a cell line cannot reliablyreflect the biology, heterogeneity, or therapeutic response of a primarybrain tumor.

[0112] Applicants obtained tumor tissue, snap frozen in the operationhall from unknown patients, which was confirmed as glioblastoma grade IVby neuropathology. These tissues served as the experimental sample.Human whole brain tissue (Clontech Laboratories, Palo Alto, USA) servedas control sample. Poly-A⁺ RNA prepared from the cells was convertedinto double-stranded cDNA (dscDNA).

[0113] Briefly, the ds-cDNA's from control and disease states weresubjected to kinetic re-annealing hybridization during whichnormalization of transcript abundances and enrichment for differentiallyexpressed transcripts (i.e., subtraction) occurs. Normalized-subtractedds-cDNAs were cloned into a plasmid vector, a large number ofrecombinant bacterial clones were picked, and their recombinant insertswere isolated by PCR. High-density cDNA arrays of those PCR productswere screened with cDNA probes derived from the original control anddisease states. Thus, only clones displaying a significanttranscriptional induction and/or repression were sequenced and carriedforward for massive expression profiling using a variety of temporal,spatial and disease-related probe sets.

[0114] The selected PCR products (fragments of 200-2000 bp in size) fromclones showing a significant transcriptional induction and/or repressionwere sequenced and functionally annotated in AGY's proprietary databasestructure (See WO01/13105). Because large sequence fragments wereutilized in the sequencing step, the data generated has a much higherfidelity and specificity than other approaches, such as SAGE. Theresulting sequence information was compared to public databases usingthe BLAST (blastn) and tblastx algorithm. The results are listed inTable 1, below: TABLE 1 RELATIVE EXPRESSION NUMBER OF CLONES PROTEINLEVEL ISOLATED (out of 20,000) ARP2 ˜2 times 13 SPARC ˜2-5.6 times 100CMET ˜1.2-2.5 times 30 CD 44 ˜2.3-3.0 times 6 BEHAB ˜2-6 times 180TSPAN3 ˜2.0-3.0 times 7 VIPR2 ˜3.0 times 3 OPN ˜2.0-3.0 times 19 PTN˜˜1.8-2.6 times 26 PTPζ ˜2.0-4.0 times 20

[0115] As one of skill in the art will appreciate from this data, eachof these proteins is individually useful as a target for the treatmentand/or imaging of brain tumors.

[0116] Characteristics of Protein Targets Utilized in the Invention

[0117] ARP2

[0118] Given the experiments described above, and the results of Table1, ARP-2 was selected as a target for selective immuno-therapeuticagents in targeting and/or imaging brain tumors. The mature proteinconsists of 493 amino acids and contains two potential consensusglycosylation sites. The complete cDNA sequence encoding ARP-2 isprovided in SEQ ID NO. 7, and the complete amino acid sequence of ARP-2is provided in SEQ ID NO. 8. ARP-2 is a 64 kDa, single chain, acidic,angiopoeitin-like protein that includes multiple functional domains,such as a hydrophobic signal sequence from amino acids 1-21 (which istypical of secreted proteins), a coiled-coil domain at the aminoterminal end from approximately amino acid sequences 22-274, and afibrinogen-like domain, from approximately about residues 275 through493. Two major isoforms have been observed, one 2.4 Kb in size and theother about 4 Kb. Both forms are abundant in heart, small intestine,spleen and stomach.

[0119] As used herein, a compound that specifically binds to ARP-2 isany compound (such as an antibody) that has a binding affinity for anynaturally occurring isoform, splice variant, or polymorphism of ARP-2,explicitly including the isoforms described herein. As one of ordinaryskill in the art will appreciate, such “specific” binding compounds(e.g., antibodies) may also bind to other closely related proteins thatexhibit significant homology (such as greater than 90% identity, morepreferably greater than 95% identity, and most preferably greater than99% identity) with the amino acid sequence of ARP-2. Such proteinsinclude truncated forms or domains of ARP-2, and recombinantlyengineered alterations of ARP-2. For example, a portion of SEQ ID NO. 8may be engineered to include a non-naturally occurring cysteine forcross linking to an immunoconjugate protein, as described.

[0120] In general, it is preferred that the antibodies utilized in thecompositions and methods of the invention bind to the fibrinogen domainbut need not be restricted to this domain. The antibody may bind to theextracellular region of ARP-2. It is to be noted that antibodies whichbind to this secreted protein are useful in the invention as cytotoxicdelivery agents, as well as functional inhibition agents, as one ofordinary skill would expect that the concentration of ARP-2 would beincreased adjacent the tumor cells which, due to the need forvascularization, over-express the protein.

[0121] When raising antibodies to ARP-2, the entire protein (either theunsecreted precursor or the secreted protein), or a portion thereof, maybe utilized. For instance, the carboxyl-terminal fibrinogen like domain,or any portion of the amino-terminal coiled-coil domain may be utilized.For instance, amino acids 22-274, which make up the fibrinogen likedomain, may be used. Larger ARP-2 proteins and domains may be producedutilizing any suitable recombinant vector/protein production system,such as the baculovirus transfection system outlined below, after beingamplified from a fetal brain cDNA library (as available from, e.g.,Clontech, Palo Alto, Calif.) or another suitable source.

[0122] When utilizing an entire protein, or a larger section of theprotein, antibodies may be raised by immunizing the production animalwith the protein and a suitable adjuvant (e.g., Fruend's, Fruend'scomplete, oil-in-water emulsions, etc.). In these cases, the ARP-2protein (or a portion thereof) can serve as the ARP-2 antigen. When asmaller peptide is utilized, it is advantageous to conjugate the peptidewith a larger molecule to make an immunostimulatory conjugate for use asthe ARP-2 antigen. Commonly utilized conjugate proteins that arecommercially available for such use include bovine serum albumin (BSA)and keyhole limpet hemocyanin (KLH). In order to raise antibodies toparticular epitopes, peptides derived from the full ARP-2 sequence maybe utilized. Preferably, one or more 8-30 aa peptide portions of the ECdomain of ARP-2 are utilized, with peptides in the range of 10-20 beinga more economical choice. Custom-synthesized peptides in this range areavailable from a multitude of vendors, and can be order conjugated toKLH or BSA. Alternatively, peptides in excess of 30 amino acids may besynthesized by solid-phase methods, or may be recombinantly produced ina suitable recombinant protein production system. In order to ensureproper protein glycosylation and processing, an animal cell system(e.g., Sf9 or other insect cells, CHO or other mammalian cells) ispreferred. Other information useful in designing an antigen for theproduction of antibodies to ARP-2 may be deduced by those of skill inthe art by homology analysis of SEQ ID NO. 8.

[0123] The fibrinogen domain of human ARP-2 is hypothesized to interactwith one or more an unknown receptor for the purposes of angiogenesis.The interaction of ARP-2 to these molecules may be through either of theaforementioned structural motifs. Thus, in alternative embodiments ofthe compositions and methods of the invention, antibody moieties areutilized which bind to ARP-2 at a site on the protein that alters thebinding of an extracellular molecule to ARP-2. Such ARP-2 activityaltering antibodies may be utilized in therapeutic compositions in anunconjugated form (e.g., the antibody in an acceptable pharmaceuticalcarrier), or may be conjugated to either a therapeutic moiety (creatinga double-acting therapeutic agent) or an imaging moiety (creating a dueltherapeutic/imaging agent).

[0124] Selection of antibodies which alter (enhance or inhibit) thebinding of a ARP-2 to a receptor may be accomplished by astraightforward binding inhibition/enhancement assay. According tostandard techniques, the binding of a labeled (e.g., fluorescently orenzyme-labeled) antibody to ARP-2, which has been immobilized in amicrotiter well, is assayed in both the presence and absence of theappropriate ligand. The change in binding is indicative of either anenhancer (increased binding) or competitive inhibitor (decreasedbinding) relationship between the antibody and the ligand. Such assaysmay be carried out in high-throughput formats (e.g., 384 well plateformats, in robotic systems) for the automated selection of monoclonalantibody candidates for use as ARP-2 ligand-binding inhibitors orenhancers.

[0125] In addition, antibodies which are useful for altering thefunction of ARP-2 may be assayed in functional formats, such asendothelial sprouting assays and cell migration assays described in theexamples. Thus, antibodies that exhibit the appropriate anti-tumoreffect may be selected without direct knowledge of a binding ligand.

[0126] SPARC

[0127] Given the experiments described above, and the results of Table1, SPARC was selected as a target for selective immuno-therapeuticagents in targeting and/or imaging brain tumors. The mature proteinconsists of 286 amino acids (after cleavage of the signal peptide) andcontains two potential Asn-X-Thr/Ser N-glycosylation sites, located atpositions 71 and 99 of the mature protein. The complete cDNA sequenceencoding SPARC is provided in SEQ ID NO. 9, and the complete amino acidsequence of SPARC is provided in SEQ ID NO. 10. SPARC is an abundant 33kDa, single chain, acidic, extracellular calcium binding protein thatcontains a flexible N-terminal acidic domain I (˜50 amino acids), afollistatin-like (FS) domain (˜75 residues), and a C-terminalextracellular calcium-binding (EC) domain with a pair of EF-hand loops(˜150 residues). The N-terminal domain shows a low affinity Ca2+ bindingsite, a transglutaminase cross linking site, and inhibits cell spreadingin cell culture assays. Calcium-dependent binding of SPARC to the triplehelix of several fibrillar collagen types and basement membrane collagentype IV has been mapped to the EC domain. Two isoforms have beendescribed, bone SPARC with a molecular weight of 31,000 kDa and plateletSPARC with a molecular weight of 33,000 kDa.

[0128] As used herein, a compound that specifically binds to SPARC isany compound (such as an antibody) that has a binding affinity for anynaturally occurring isoform, splice variant, or polymorphism of SPARC,explicitly including the isoforms described herein. As one of ordinaryskill in the art will appreciate, such “specific” binding compounds(e.g., antibodies) may also bind to other closely related proteins thatexhibit significant homology (such as greater than 90% identity, morepreferably greater than 95% identity, and most preferably greater than99% identity) with the amino acid sequence of SPARC. Such proteinsinclude truncated forms or domains of SPARC, and recombinantlyengineered alterations of SPARC. For example, a portion of SEQ ID NO. 10may be engineered to include a non-naturally occurring cysteine forcross linking to an immunoconjugate protein, as described.

[0129] In general, it is preferred that the antibodies utilized in thecompositions and methods of the invention bind to the extracellulardomain (amino acids 130-280). It is preferable that this binding inhibitthe activity of SPARC. The antibody may bind to the EF hand which isknown to bind Ca2+ with high affinity, but need not be restricted tothis domain. It is to be noted that antibodies which bind to SPARC areuseful in both cytotoxic and imaging embodiments of the invention, asone of ordinary skill would expect that the concentration of SPARC inthe extracellular matrix would be increased around tumor cells whichover-express the protein.

[0130] When raising antibodies to SPARC the entire protein (either theunsecreted precursor or the secreted protein), or a portion thereof, maybe utilized. For instance, the C terminal extracellular (EC) domain, orany portion of the flexible N-terminal domain I, or FS domain may beutilized. For instance, amino acids 125-275, which make up the ECdomain, may be used. Larger SPARC proteins and domains may be producedutilizing any suitable recombinant vector/protein production system,such as the baculovirus transfection system outlined below, after beingamplified from a fetal brain cDNA library (as available from, e.g.,Clontech, Palo Alto, Calif.) or another suitable source.

[0131] When utilizing an entire protein, or a larger section of theprotein, antibodies may be raised by immunizing the production animalwith the protein and a suitable adjuvant (e.g., Fruend's, Fruend'scomplete, oil-in-water emulsions, etc.). In these cases, the SPARCprotein (or a portion thereof) can serve as the SPARC antigen. When asmaller peptide is utilized, it is advantageous to conjugate the peptidewith a larger molecule to make an immunostimulatory conjugate for use asthe SPARC antigen. Commonly utilized conjugate proteins that arecommercially available for such use include bovine serum albumin (BSA)and keyhole limpet hemocyanin (KLH). In order to raise antibodies toparticular epitopes, peptides derived from the full SPARC sequence maybe utilized. Preferably, one or more 8-30 aa peptide portions of the ECdomain of SPARC are utilized, with peptides in the range of 10-20 beinga more economical choice. Custom-synthesized peptides in this range areavailable from a multitude of vendors, and can be order conjugated toKLH or BSA. Alternatively, peptides in excess of 30 amino acids may besynthesized by solid-phase methods, or may be recombinantly produced ina suitable recombinant protein production system. In order to ensureproper protein glycosylation and processing, an animal cell system(e.g., Sf9 or other insect cells, CHO or other mammalian cells) ispreferred. Other information useful in designing an antigen for theproduction of antibodies to SPARC, including glycosylation sites, isprovided in SEQ ID NO. 10.

[0132] The EC domain of human SPARC is known to interact with thecollagens I, III, IV and V, and to bind to vitronectin, all of which arecomponents of the extracellular matrix surrounding gliomas. The bindingof SPARC to these molecules may play a significant role in theoncogenesis and growth of neoplastic cells in the brain. Thus, inalternative embodiments of the compositions and methods of theinvention, antibody moieties are utilized which bind to SPARC at a siteon the protein that alters the binding of an extracellular molecule,such as an ECM molecule, to SPARC. Such SPARC activity alteringantibodies may be utilized in therapeutic compositions in anunconjugated form (e.g., the antibody in an acceptable pharmaceuticalcarrier), or may be conjugated to either a therapeutic moiety (creatinga double-acting therapeutic agent) or an imaging moiety (creating a dueltherapeutic/imaging agent).

[0133] Selection of antibodies which alter (enhance or inhibit) thebinding of a ligand to SPARC may be accomplished by a straightforwardbinding inhibition/enhancement assay. According to standard techniques,the binding of a labeled (e.g., fluorescently or enzyme-labeled)antibody to SPARC, which has been immobilized in a microtiter well, isassayed in both the presence and absence of the appropriate ligand. Thechange in binding is indicative of either an enhancer (increasedbinding) or competitive inhibitor (decreased binding) relationshipbetween the antibody and the ligand. Such assays may be carried out inhigh-throughput formats (e.g., 384 well plate formats, in roboticsystems) for the automated selection of monoclonal antibody candidatesfor use as SPARC ligand-binding inhibitors or enhancers.

[0134] In addition, antibodies which are useful for altering thefunction of SPARC may be assayed in functional formats, such as theHUVEC tube assay and cell migration assay. Thus, antibodies that exhibitthe appropriate anti-tumor effect may be selected without directknowledge of a binding ligand or molecular function.

[0135] c-MET

[0136] Given the experiments described above, and the results of Table1, c-MET was selected as a target for selective immuno-therapeuticagents in targeting and/or imaging brain tumors. The complete cDNAsequence encoding c-MET is provided in SEQ ID NO. 11, and the completeamino acid sequence of c-MET is provided in SEQ ID NO. 12. c-MET is atype I membrane protein heterodimer. Generally, two different receptorvariants originate by post-translational processing of a commonsinge-chain precursor of 170 kDa. Isoform p190MET is formed of a 50 kDaα-chain and a 145 kDa α-chain that are disulfide linked, and isoformp140Met is formed of a 50 kDa α-chain and an 85 kDa β-chain, lacking thecytoplasmic kinase domain. This 85 kDa β chain is likely atrans-membrane glycoprotein that is bound to the cell surface. Truncatedforms of c-MET containing the 50 kDa α-chain and a carboxyl-terminallytruncated 75 kDa β sub-unit have also been described. The 75 kDa formarises by post-translational proteolytic processing, lacks thetrans-membrane domain, and is secreted from the cell.

[0137] As used herein, a compound that specifically binds to c-MET isany compound (such as an antibody) that has a binding affinity for anynaturally occurring isoform, splice variant, or polymorphism of c-MET,explicitly including the three isoforms described herein. As one ofordinary skill in the art will appreciate, such “specific” bindingcompounds (e.g., antibodies) may also bind to other closely relatedproteins that exhibit significant homology (such as greater than 90%identity, more preferably greater than 95% identity, and most preferablygreater then 99% identity) with the amino acid sequence of c-MET. Suchproteins include truncated forms or domains of c-MET, and recombinantlyengineered alterations of c-MET. For example, a portion of SEQ ID NO. 12may be engineered to include a non-naturally occurring cysteine forcross-linking to an immunoconjugate protein, as described below.

[0138] In general, it is preferred that the antibodies utilized in thecompositions and methods of the invention bind to the membrane-boundisoforms of the protein, as this will more specifically target thecytotoxic therapeutic agent, or the imaging agent, to the brain tumorcell. However, embodiments that utilize antibodies that bind to thesecreted isoform of the protein are also useful in the invention, as oneof ordinary skill would expect that the concentration of the secretedisoform would also be increased adjacent to brain tumor cells whichover-express the protein.

[0139] The amino acid sequence of full length c-MET consists of 1408amino acids, as the sequence was first deduced by Park et al.,(“Sequence of MET proto-oncogene cDNA has features characteristic of thetyrosine kinase family of growth-factor receptors” Proc. nat. Acad. Sci.U.S.A. 84:6379-6383 (1987)) and 1390 amino acids, as later deduced byPrat et al. (“C-terminal truncated forms of Met, the Hepatocyte GrowthFactor” Mol Cell. Biol. 11:5954-5962 (1991)). According to Prat et al.,the first N-terminal amino acids 1-24 of SEQ ID NO. B′ [B′] are for themost part hydrophobic, and could serve as a signal sequence fortransporting the protein into the lumen of the endoplasmic reticulum.The α chain makes up the extracellular domain of the mature c-METprotein and spans amino acids 24-306 of SEQ ID NO. 12. The β chain wouldconsist of 1,084-5 amino acids with the predicted β chain extracellulardomain being amino acids 306 to 932, the single transmembranehydrophobic segment being amino acids 933 to 955, and the intracellulardomain being amino acids 956 to 1390 of SEQ ID NO 12.

[0140] When raising antibodies to c-MET, the entire protein, a dimericsubunit, or a portion thereof may be utilized. For instance, theextracellular domain of the α or β sub-units or the secreted orextracellular portion of the truncated forms may be utilized. Forinstance, amino acids that constitute the α sub-unit, amino acids24-306, may be used. Larger c-MET proteins and domains may be producedutilizing any suitable recombinant vector/protein production system,such as the baculovirus transfection system outlined below, after beingamplified from a fetal brain cDNA library (as available from, e.g.,Clontech, Palo Alto, Calif.) or another suitable source.

[0141] When utilizing an entire protein, or a larger section of theprotein, antibodies may be raised by immunizing the production animalwith the protein and a suitable adjuvant (e.g., Fruend's, Fruend'scomplete, oil-in-water emulsions, etc.). In these cases, the c-METprotein (or a portion thereof) can serve as the c-MET antigen. When asmaller peptide is utilized, it is advantageous to conjugate the peptidewith a larger molecule to make an immunostimulatory conjugate for use asthe c-MET antigen. Commonly utilized conjugate proteins that arecommercially available for such use include bovine serum albumin (BSA)and keyhole limpet hemocyanin (KLH). In order to raise antibodies toparticular epitopes, peptides derived from the full c-MET sequence maybe utilized. Preferably, one or more 8-30 amino acid peptide portions ofan extracellular domain of c-MET are utilized, with peptides in therange of 10-20 being a more economical choice. Custom-synthesizedpeptides in this range are available from a multitude of vendors, andcan be order conjugated to KLH or BSA. Alternatively, peptides in excessof 30 amino acids may be synthesized by solid-phase methods, or may berecombinantly produced in a suitable recombinant protein productionsystem. In order to ensure proper protein glycosylation and processing,an animal cell system (e.g., Sf9 or other insect cells, CHO or othermammalian cells) is preferred. Other information useful in designing anantigen for the production of antibodies to c-MET, includingglycosylation sites, is provided in SEQ ID NO. 12.

[0142] The extracellular domain of human c-MET binds hepatocyte growthfactor (HGF).

[0143] Because HGF is largely expressed in mesenchymal andneuroectodermal tissues and released to the extracellular compartment,paracrine and/or autocrine signaling implicate tumor genesis inmesenchymal and neuroectodermal tumors and other tumor cells that overexpress the c-MET receptor. Recent studies have shown that the c-METproto-oncogene is frequently overexpressed in many types of epithelialtumors, in spontaneously transformed NIH/3T3 fibroblasts, and inperipheral nerve sheath tumors. In alternative embodiments of thecompositions and methods of the invention, antibody moieties areutilized which bind to c-MET at a site on the protein which alters thebinding of an extracellular ligand molecule, such as HGF, to c-MET. Suchc-MET activity altering antibodies may be utilized in therapeuticcompositions in an unconjugated form (e.g., the antibody in anacceptable pharmaceutical carrier), or may be conjugated to either atherapeutic moiety (creating a double-acting therapeutic agent) or animaging moiety (creating a duel therapeutic/imaging agent).

[0144] Selection of antibodies that alter (enhance or inhibit) thebinding of a ligand to c-MET may be accomplished by a straightforwardbinding inhibition/enhancement assay. According to standard techniques,the binding of a labeled (e.g., fluorescently or enzyme-labeled)antibody to c-MET, which has been immobilized in a microtiter well, isassayed in both the presence and absence of the ligand. The change inbinding is indicative of either an enhancer (increased binding) orcompetitive inhibitor (decreased binding) relationship between theantibody and the ligand. Such assays may be carried out inhigh-throughput formats (e.g., 384 well plate formats, in roboticsystems) for the automated selection of monoclonal antibody candidatesfor use as c-MET ligand-binding inhibitors or enhancers.

[0145] In addition, antibodies that are useful for altering the functionof c-MET may be assayed in functional formats, such as the endothelialsprouting assay and cell migration assay. Thus, antibodies which exhibitthe appropriate anti-tumor effect may be selected without directknowledge of a molecular function.

[0146] BEHAB

[0147] Given the experiments described above, and the results of Table1, BEHAB was selected as a target for selective immuno-therapeuticagents in targeting and/or imaging brain tumors. The complete cDNAsequence encoding BEHAB GPI isoform is provided in SEQ ID NO. 13, andthe complete amino acid sequence of this BEHAB isoform is provided inSEQ ID NO. 14. Two isoforms have been isolated to date: a full-lengthisoform that is secreted into the extracellular matrix and a shorterisoform that has a hydrophobic carboxy terminus instead of the typicallectican carboxyl terminus, which predicts a glycophosphatidylinositol(GPI) anchor. BEHAB contains an N-terminal hyaluronan (HA)-bindingdomain, which comprises an immunoglobulin-like loop and two proteoglycantandem repeats, a C-terminal epidermal growth factor (EGF)-like repeat,a C-type lectin-like domain, and a complement regulatory protein(CRP)-like domain. The central region of the protein contains sites forglycosylation and proteolytic cleavage (between glu395-Ser396 of themature protein, after signal peptide cleavage) by metallo-protease. Thecomplete cDNA of the secreted isoform is 2878 bp encoding 912 aminoacids of 99 kDa. The GPI isoform, for which sequences SEQ ID NO. 13 andSEQ ID NO. 14 are given, is 2558 bp encoding 672 amino acids of 72 kDa.The GPI-linked form is generated by a ‘no splice’ event, with thetranscript reading through an exon/intron junction thereby extending theopen reading frame to a stop codon 74 nucleotides further downstream.

[0148] As used herein, a compound that specifically binds to BEHAB isany compound (such as an antibody) that has a binding affinity for anynaturally occurring isoform, splice variant, or polymorphism of BEHAB,explicitly including the two splice variants described herein. As one ofordinary skill in the art will appreciate, such “specific” bindingcompounds (e.g., antibodies) may also bind to other closely relatedproteins that exhibit significant homology (such as greater than 90%identity, more preferably greater than 95% identity, and most preferablygreater then 99% identity) with the amino acid sequence of BEHAB. Suchproteins include truncated forms or domains of BEHAB, and recombinantlyengineered alterations of BEHAB. For example, a portion of SEQ ID NO. 14may be engineered to include a non-naturally occurring cysteine forcross-linking to an immunoconjugate protein, as described below.

[0149] In general, it is preferred that the antibodies utilized in thecompositions and methods of the invention bind to the membrane-boundisoform of the protein, as this will more specifically target thecytotoxic therapeutic agent, or the imaging agent, to the brain tumorcell. However, embodiments that utilize antibodies that bind to thesecreted isoform of the protein are also useful in the invention, as oneof ordinary skill would expect that the concentration of the secretedisoform would also be increased adjacent to brain tumor cells whichover-express the protein.

[0150] When raising antibodies to BEHAB, the entire protein, or aportion thereof, may be utilized. For instance, any one of theaforementioned domains of the secreted protein or an extracellularportion of the truncated, membrane bound GPI form may be utilized. Forinstance, amino acids that constitute the hyaluronic acid bindingdomain, amino acids 44-247, which includes the Ig like domain at aminoacids 44-140, may be used. Larger BEHAB proteins and domains may beproduced utilizing any suitable recombinant vector/protein productionsystem, such as the baculovirus transfection system outlined below,after being amplified from a fetal brain cDNA library (as availablefrom, e.g., Clontech, Palo alto, CA) or another suitable source.

[0151] When utilizing an entire protein, or a larger section of theprotein, antibodies may be raised by immunizing the production animalwith the protein and a suitable adjuvant (e.g., Fruend's, Fruend'scomplete, oil-in-water emulsions, etc.). In these cases, the Brevicanprotein (or a portion thereof) can serve as the BEHAB antigen. When asmaller peptide is utilized, it is advantageous to conjugate the peptidewith a larger molecule to make an immunostimulatory conjugate for use asthe BEHAB antigen. Commonly utilized conjugate proteins that arecommercially available for such use include bovine serum albumin (BSA)and keyhole limpet hemocyanin (KLH). In order to raise antibodies toparticular epitopes, peptides derived from the full Brevican sequencemay be utilized. Preferably, one or more 8-30 amino acid peptideportions of an extracellular domain of BEHAB are utilized, with peptidesin the range of 10-20 being a more economical choice. Custom-synthesizedpeptides in this range are available from a multitude of vendors, andcan be order conjugated to KLH or BSA. Alternatively, peptides in excessof 30 amino acids may be synthesized by solid-phase methods, or may berecombinantly produced in a suitable recombinant protein productionsystem. In order to ensure proper protein glycosylation and processing,an animal cell system (e.g., Sf9 or other insect cells, CHO or othermammalian cells) is preferred.

[0152] The hyaluronic acid binding domain of human BEHAB binds tohyaluronic acid (HA). Because HA is largely expressed in the ECMsurrounding gliomas and because recent studies have shown that the BEHABprotein is frequently overexpressed in primary brain tumors, it issuggested that the up-regulation of BEHAB may be a crucial step inreturning the unmalleable mature extracellular matrix to a more immaturematrix, permissive for cell growth, thereby promoting the progression ofprimary brain tumors. Thus, in alternative embodiments of thecompositions and methods of the invention, antibody moieties areutilized which bind to BEHAB at a site on the protein which alters thebinding of an extracellular ligand molecule (e.g., HA) to BEHAB. SuchBEHAB activity altering antibodies may be utilized in therapeuticcompositions in an unconjugated form (e.g., the antibody in anacceptable pharmaceutical carrier), or may be conjugated to either atherapeutic moiety (creating a double-acting therapeutic agent) or animaging moiety (creating a duel therapeutic/imaging agent).

[0153] Selection of antibodies that alter (enhance or inhibit) thebinding of a ligand to BEHAB may be accomplished by a straightforwardbinding inhibition/enhancement assay. According to standard techniques,the binding of a labeled (e.g., fluorescently or enzyme-labeled)antibody to BEHAB, which has been immobilized in a microtiter well, isassayed in both the presence and absence of the ligand. The change inbinding is indicative of either an enhancer (increased binding) orcompetitive inhibitor (decreased binding) relationship between theantibody and the ligand. Such assays may be carried out inhigh-throughput formats (e.g., 384 well plate formats, in roboticsystems) for the automated selection of monoclonal antibody candidatesfor use as BEHAB ligand-binding inhibitors or enhancers.

[0154] In addition, antibodies that are useful for altering the functionof BEHAB may be assayed in functional formats, such as the HUVEC tubeassay and the cell migration assay described below. Thus, antibodieswhich exhibit the appropriate anti-tumor effect may be selected withoutdirect knowledge of molecular function.

[0155] CD-44

[0156] Given the experiments described above, and the results of Table1, CD-44 was selected as a target for selective immuno-therapeuticagents in targeting and/or imaging brain tumors. The complete cDNAsequence encoding CD-44E is provided in SEQ ID NO. 15, and the completeamino acid sequence of CD-44, indicating various splicing variationlocations, is provided in SEQ ID NO. 16. CD-44 is a proteoglycan that isexpressed as two major splice variants. CD-44E is a 150 kDa proteinisolated from epithelial cells. CD-44E has a C-terminal cytoplasmictail, a hydrophobic transmembrane domain of 23 amino acids, and anN-terminal extracellular region of 248 amino acids. The extracellulardomain is O-glycosylated and also binds chondroitin sulfate. Inaddition, CD-44E it has two of the three immunodominant epitope clustersof native gp90Hermes. CD-44E contains an additional 132 amino acids inthe extracellular region. and CD-44H is a 90 kDa protein isolated fromhematopoietic cells. In addition, CD-44R1 and CD-44R2 are 2 isoformsexpressed by hematopoietic cells. The complete cDNA sequence of the 90kDa CD-44H isoform consist of 1795 bps, encoding a 341 amino acidprotein.

[0157] As used herein, a compound that specifically binds to CD-44 isany compound (such as an antibody) that has a binding affinity for anynaturally occurring isoform, splice variant, or polymorphism of CD-44,explicitly including the isoforms described herein. As one of ordinaryskill in the art will appreciate, such “specific” binding compounds(e.g., antibodies) may also bind to other closely related proteins thatexhibit significant homology (such as greater than 90% identity, morepreferably greater than 95% identity, and most preferably greater then99% identity) with the amino acid sequence of CD-44. Such proteinsinclude truncated forms or domains of CD-44, and recombinantlyengineered alterations of CD-44. For example, a portion of SEQ ID NO. 16may be engineered to include a non-naturally occurring cysteine forcross-linking to an immunoconjugate protein, as described below.

[0158] According to the human full length, CD-44H protein has an overallprimary structure of 90 kDa, which consist of 341 amino acids. TheN-terminus is located outside of the cell and the extracellular domainconsist of 248 amino acids. The C-terminus is located inside of the celland the intracellular domain consist of 72 amino acids, while thetransmembrane region consist of 21 amino acids. The CD-44 gene contains20 exons, of which exons 1-5, 15-17 and 19 encode the CD44H isoform. Theintervening exons 6, 6a, 7-14 (also designated v1-v10) are alternativelyspliced to generate the variant isoforms with an insertion at themembrane proximal region of the extracellular domain between amino acids202 and 203. See Bajorath (2000). Proteins: structure, function, andgenetic, 39:103-111; and Ilangumaram et al. Leukemia and Lymphoma,35:455-469.

[0159] When raising antibodies to CD-44, the entire protein, or aportion thereof, may be utilized. For instance, any portion of theextracellular domain may be utilized. For instance, the amino acidsbetween the signal sequence and amino acid 202 may be used. Larger CD-44proteins and domains may be produced utilizing any suitable recombinantvector/protein production system, such as the baculovirus transfectionsystem outlined below, after being amplified from a fetal brain cDNAlibrary (as available from, e.g., Clontech, Palo Alto, Calif.) oranother suitable source.

[0160] When utilizing an entire protein, or a larger section of theprotein, antibodies may be raised by immunizing the production animalwith the protein and a suitable adjuvant (e.g., Fruend's, Fruend'scomplete, oil-in-water emulsions, etc.). In these cases, the CD-44protein (or a portion thereof) can serve as the CD-44 antigen. When asmaller peptide is utilized, it is advantageous to conjugate the peptidewith a larger molecule to make an immunostimulatory conjugate for use asthe CD-44 antigen. Commonly utilized conjugate proteins that arecommercially available for such use include bovine serum albumin (BSA)and keyhole limpet hemocyanin (KLH). In order to raise antibodies toparticular epitopes, peptides derived from the full CD-44 sequence maybe utilized. Preferably, one or more 8-30 amino acid peptide portions ofan extracellular domain of CD-44 are utilized, with peptides in therange of 10-20 being a more economical choice. Custom-synthesizedpeptides in this range are available from a multitude of vendors, andcan be order conjugated to KLH or BSA. Alternatively, peptides in excessof 30 amino acids may be synthesized by solid-phase methods, or may berecombinantly produced in a suitable recombinant protein productionsystem. In order to ensure proper protein glycosylation and processing,an animal cell system (e.g., Sf9 or other insect cells, CHO or othermammalian cells) is preferred. Other information useful in designing anantigen for the production of antibodies to CD-44, includingglycosylation sites, is provided in SEQ ID NO. D′.

[0161] Hyaluronan (HA) is a polymeric glycosaminoglycan and a majorcomponent of the extracellular matrix. CD-44 is one of the principalreceptors for HA. Within the normal CNS, the CD-44 protein has beenlocalized to astrocytes in the white matter. CD-44H has been shown to bethe predominant isoform in normal brain and neuroectoderm-derivedtumors. Hence, the up-regulation of CD-44 may be a crucial step in braintumor invasiveness and migration. Thus, in alternative embodiments ofthe compositions and methods of the invention, antibody moieties areutilized which bind to CD-44 at a site on the protein which alters thebinding of an extracellular ligand molecule (e.g., HA) to CD-44. SuchCD-44 activity altering antibodies may be utilized in therapeuticcompositions in an unconjugated form (e.g., the antibody in anacceptable pharmaceutical carrier), or may be conjugated to either atherapeutic moiety (creating a double-acting therapeutic agent) or animaging moiety (creating a duel therapeutic/imaging agent).

[0162] Selection of antibodies that alter (enhance or inhibit) thebinding of a ligand to CD-44 may be accomplished by a straightforwardbinding inhibition/enhancement assay. According to standard techniques,the binding of a labeled (e.g., fluorescently or enzyme-labeled)antibody to CD-44, which has been immobilized in a microtiter well, isassayed in both the presence and absence of the ligand. The change inbinding is indicative of either an enhancer (increased binding) orcompetitive inhibitor (decreased binding) relationship between theantibody and the ligand. Such assays may be carried out inhigh-throughput formats (e.g., 384 well plate formats, in roboticsystems) for the automated selection of monoclonal antibody candidatesfor use as CD-44 ligand-binding inhibitors or enhancers.

[0163] In addition, antibodies that are useful for altering the functionof CD-44 may be assayed in functional formats, such as endothelialsprouting assay and cell migration assay. Thus, antibodies which exhibitthe appropriate anti-tumor effect may be selected without directknowledge of molecular function.

[0164] TSPAN3

[0165] Given the experiments described above, and the results of Table1, TSPAN3 was selected as a target for selective immuno-therapeuticagents in targeting and/or imaging brain tumors. The complete cDNAsequence encoding TSPAN3 is provided in SEQ ID NO. 17, and the completeamino acid sequence of TSPAN3 is provided in SEQ ID NO. 18. Tetraspaninis a 253 amino acid membrane bound protein. No isoforms have beenisolated to date. TSPAN3, as is characteristic of the tetraspaninfamily, contains four transmembrane domains, putatively comprising aminoacids 12-32, 51-71, 86-106, and 213-233. The protein has two putativeextracellular domains, amino acids 33-50 and 107-212, and three putativecytoplasmic domains, amino acids 1-11, 72-85, and 234-235. PutativeN-linked glycosylation sites are listed in SEQ ID NO. 18.

[0166] As used herein, a compound that specifically binds to TSPAN3 isany compound (such as an antibody) that has a binding affinity for anynaturally occurring isoform, splice variant, or polymorphism of TSPAN3.As one of ordinary skill in the art will appreciate, such “specific”binding compounds (e.g., antibodies) may also bind to other closelyrelated proteins that exhibit significant homology (such as greater than90% identity, more preferably greater than 95% identity, and mostpreferably greater then 99% identity) with the amino acid sequence ofTSPAN3. Such proteins include truncated forms or domains of TSPAN3, andrecombinantly engineered alterations of TSPAN3. For example, a portionof SEQ ID NO. 18 may be engineered to include a non-naturally occurringcysteine for cross-linking to an immunoconjugate protein, as describedbelow.

[0167] In general, it is preferred that the antibodies utilized in thecompositions and methods of the invention bind to the membrane-boundisoform of the protein, as this will more specifically target thecytotoxic therapeutic agent, or the imaging agent, to the brain tumorcell. The only currently known form of TSPAN3 is membrane-bound.However, embodiments that utilize antibodies that bind to any secretedisoform of the protein are also useful in the invention, as one ofordinary skill would expect that the concentration of the secretedisoform would also be increased adjacent to brain tumor cells whichover-express the protein. Likewise, it is preferred that the antibodiesutilized in the invention bind to an extracellular domain of theprotein, as are described in the SEQ ID NO. 18. The cysteine residues atpositions 147, 148, and 197 of SEQ ID NO. 18 in the second extracellulardomain are highly conserved among the tetraspanin family and are thoughtto be essential for proper tetraspanin function. Thus, in some preferredembodiments of the invention, the antibodies utilized in the inventionbind to an epitope comprising, or alternatively very near to, one ofthese cysteine residues.

[0168] When raising antibodies to TSPAN3, the entire protein, or aportion thereof, may be utilized. For instance, any one of theaforementioned domains of the secreted protein or an extracellularportion of the truncated, membrane bound GPI form may be utilized. Forinstance, amino acids that constitute one of the extracellular domains,amino acids 33-50 or 107-212, may be used. Larger TSPAN3 proteins anddomains may be produced utilizing any suitable recombinantvector/protein production system, such as the baculovirus transfectionsystem outlined below, after being amplified from a fetal brain cDNAlibrary (as available from, e.g., Clontech, Palo Alto, Calif.) oranother suitable source.

[0169] When utilizing an entire protein, or a larger section of theprotein, antibodies may be raised by immunizing the production animalwith the protein and a suitable adjuvant (e.g., Fruend's, Fruend'scomplete, oil-in-water emulsions, etc.). In these cases, the tetraspanin3 protein (or a portion thereof) can serve as the TSPAN3 antigen. When asmaller peptide is utilized, it is advantageous to conjugate the peptidewith a larger molecule to make an immunostimulatory conjugate for use asthe TSPAN3 antigen. Commonly utilized conjugate proteins that arecommercially available for such use include bovine serum albumin (BSA)and keyhole limpet hemocyanin (KLH). In order to raise antibodies toparticular epitopes, peptides derived from the full Brevican sequencemay be utilized. Preferably, one or more 8-30 amino acid peptideportions of an extracellular domain of TSPAN3 are utilized, withpeptides in the range of 10-20 being a more economical choice.Custom-synthesized peptides in this range are available from a multitudeof vendors, and can be order conjugated to KLH or BSA. Alternatively,peptides in excess of 30 amino acids may be synthesized by solid-phasemethods, or may be recombinantly produced in a suitable recombinantprotein production system. In order to ensure proper proteinglycosylation and processing, an animal cell system (e.g., Sf9 or otherinsect cells, CHO or other mammalian cells) is preferred. Otherinformation useful in designing an antigen for the production ofantibodies to TSPAN3, including glycosylation sites, is provided in SEQID NO. 18.

[0170] In alternative embodiments of the compositions and methods of theinvention, antibody moieties are utilized which bind to TSPAN3 at a siteon the protein which alters the binding of an extracellular ligandmolecule to TSPAN3. Such TSPAN3 activity altering antibodies may beutilized in therapeutic compositions in an unconjugated form (e.g., theantibody in an acceptable pharmaceutical carrier), or may be conjugatedto either a therapeutic moiety (creating a double-acting therapeuticagent) or an imaging moiety (creating a duel therapeutic/imaging agent).

[0171] Selection of antibodies that alter (enhance or inhibit) thebinding of a ligand to TSPAN3 may be accomplished by a straightforwardbinding inhibition/enhancement assay. According to standard techniques,the binding of a labeled (e.g., fluorescently or enzyme-labeled)antibody to TSPAN3, which has been immobilized in a microtiter well, isassayed in both the presence and absence of the ligand. The change inbinding is indicative of either an enhancer (increased binding) orcompetitive inhibitor (decreased binding) relationship between theantibody and the ligand. Such assays may be carried out inhigh-throughput formats (e.g., 384 well plate formats, in roboticsystems) for the automated selection of monoclonal antibody candidatesfor use as TSPAN3 ligand-binding inhibitors or enhancers.

[0172] In addition, antibodies that are useful for altering the functionof TSPAN3 may be assayed in functional formats, such as the HUVEC tubeassay and the cell migration assay described below. Thus, antibodieswhich exhibit the appropriate anti-tumor effect may be selected withoutdirect knowledge of molecular function.

[0173] VIPR-2

[0174] Given the experiments described above, and the results of Table1, VIPR-2 was selected as a prime target for selectiveimmuno-therapeutic agents in targeting and/or imaging brain tumors. Thecomplete cDNA sequence encoding VIPR-2 is provided in SEQ ID NO. 19, andthe complete amino acid sequence of VIPR-2 is provided in SEQ ID NO. 20.VIPR-2 is a seven transmembrane spanning G-protein receptor. Thecomplete VIPR-2 protein is encoded by 13 exons. The initiator codon ofthe approximated 438 amino acid-encoding open reading frame is locatedin exon 1 and the termination signal is located in exon 13. The 5′untranslated region extends 187 bp upstream of the initiator codon andis extremely GC-rich (80%). The polyadenylation signal is located 2416bp downstream of the stop codon. Intron sizes range from 68 bp (intron11) to 45 bp (intron 4), the entire human gene spans 117 kb, while thecDNA sequence spans 1317 bp. Recent studies have also isolated two VIP-2receptor mRNAs of 4.6 kb and 2.3 kb in size.

[0175] As used herein, a compound that specifically binds to VIPR-2 isany compound (such as an antibody) that has a binding affinity for anynaturally occurring isoform, splice variant, or polymorphism of VIPR-2,explicitly including any isoforms described herein. As one of ordinaryskill in the art will appreciate, such “specific” binding compounds(e.g., antibodies) may also bind to other closely related proteins thatexhibit significant homology (such as greater than 90% identity, morepreferably greater than 95% identity, and most preferably greater then99% identity) with the amino acid sequence of VIPR-2. Such proteinsinclude truncated forms or domains of VIPR-2, and recombinantlyengineered alterations of VIPR-2. For example, a portion of SEQ ID NO.20 may be engineered to include a non-naturally occurring cysteine forcross-linking to an immunoconjugate protein, as described below.

[0176] In general, it is preferred that the antibodies utilized in thecompositions and methods of the invention bind to the membrane-boundisoforms of the protein, as this will more specifically target thecytotoxic therapeutic agent, or the imaging agent, to the brain tumorcell. However, embodiments that utilize antibodies that bind to thesecreted isoform of the protein are also useful in the invention, as oneof ordinary skill would expect that the concentration of the secretedisoform would also be increased adjacent to brain tumor cells whichover-express the protein.

[0177] The amino acid sequence of full length VIPR-2 consists of 437amino acids with a predicted molecular mass is 49 kDa, as the sequencewas first deduced by Lutz et al. FEBS. 334:3-8, 1993. Lutz et al.predicted that the receptor is a seven membrane spanning protein wherein the first 22 amino acids constitute a typical hydrophobic signalsequence, and the remaining amino acids constitute two membrane spanningregions between amino acids 127 to 148 and 158 to 178, two more membranespanning domains between amino acids 202 to 227 and 238 to 261, anotherbetween 278 to 303, and two final membrane spanning regions between 327to 347 and 359 to 380, with three potential N-linked glycosylation sitesfound in the amino terminal extracellular domain at residues 57, 87 and91. Sreedharan et al. describes the VIPR-2 receptor as being a 457amino-acid protein encoded by a 2.8 kb cDNA of 52 kDa. Sreedharan et al.Biochem. Biophys. Res. Commun. 203:141-148, 1994.

[0178] When raising antibodies to VIPR-2, the entire protein or aportion thereof may be utilized. For instance, the extracellular domainsof any of the seven transmembrane spanning portions of the protein maybe utilized. For instance, amino acids 179 to 201 may be used. LargerVIPR-2 proteins and domains may be produced utilizing any suitablerecombinant vector/protein production system, such as the baculovirustransfection system outlined below, after being amplified from a fetalbrain cDNA library (as available from, e.g., Clontech, Palo Alto,Calif.) or another suitable source.

[0179] When utilizing an entire protein, or a larger section of theprotein, antibodies may be raised by immunizing the production animalwith the protein and a suitable adjuvant (e.g., Fruend's, Fruend'scomplete, oil-in-water emulsions, etc.). In these cases, the VIPR-2protein (or a portion thereof) can serve as the VIPR-2 antigen. When asmaller peptide is utilized, it is advantageous to conjugate the peptidewith a larger molecule to make an immunostimulatory conjugate for use asthe VIPR-2 antigen. Commonly utilized conjugate proteins that arecommercially available for such use include bovine serum albumin (BSA)and keyhole limpet hemocyanin (KLH). In order to raise antibodies toparticular epitopes, peptides derived from the full VIPR-2 sequence maybe utilized. Preferably, one or more 8-30 amino acid peptide portions ofan extracellular domain of VIPR-2 are utilized, with peptides in therange of 10-20 being a more economical choice. Custom-synthesizedpeptides in this range are available from a multitude of vendors, andcan be order conjugated to KLH or BSA. Alternatively, peptides in excessof 30 amino acids may be synthesized by solid-phase methods, or may berecombinantly produced in a suitable recombinant protein productionsystem. In order to ensure proper protein glycosylation and processing,an animal cell system (e.g., Sf9 or other insect cells, CHO or othermammalian cells) is preferred. Other information useful in designing anantigen for the production of antibodies to VIPR-2, includingglycosylation sites, is provided in SEQ ID NO. 20.

[0180] The extracellular domain of human VIPR-2 binds PACAP-27,PACAP-38, VIP and secretin. Because these factors have been found toaffect tumor cell growth, and due to the recent discovery that theVIPR-2 receptor is overexpressed in glioblastomas (Astrocytoma gradeIV), the binding of these factors to the VIPR-2 receptor may play asignificant role in the oncogenesis and growth of astrocytoma cells inthe brain. Thus, in alternative embodiments of the of the invention,antibody moieties are utilized which bind to VIPR-2 at a site on theprotein which alters the binding of extracellular ligand molecules, suchas VIP, to VIPR-2. Such VIPR-2 activity altering antibodies may beutilized in therapeutic compositions in an unconjugated form (e.g., theantibody in an acceptable pharmaceutical carrier), or may be conjugatedto either a therapeutic moiety (creating a double-acting therapeuticagent) or an imaging moiety (creating a duel therapeutic/imaging agent).

[0181] Selection of antibodies that alter (enhance or inhibit) thebinding of a ligand to VIPR-2 may be accomplished by a straightforwardbinding inhibition/enhancement assay. According to standard techniques,the binding of a labeled (e.g., fluorescently or enzyme-labeled)antibody to VIPR-2, which has been immobilized in a microtiter well, isassayed in both the presence and absence of the ligand (e.g., vasoactiveintestinal peptide.) The change in binding is indicative of either anenhancer (increased binding) or competitive inhibitor (decreasedbinding) relationship between the antibody and the ligand. Such assaysmay be carried out in high-throughput formats (e.g., 384 well plateformats, in robotic systems) for the automated selection of monoclonalantibody candidates for use as VIPR-2 ligand-binding inhibitors orenhancers.

[0182] In addition, antibodies that are useful for altering the functionof VIPR-2 may be assayed in functional formats, such as the HUVEC tubeassay and cell migration assay. Thus, antibodies which exhibit theappropriate anti-tumor effect may be selected without direct knowledgeof a binding ligand.

[0183] PTN

[0184] Given the experiments described above, and the results of Table1, PTN was selected as a target for selective immuno-therapeutic agentsin targeting and/or imaging brain tumors. The entire PTN gene spans 65kb and 7 exons, while the mature protein is approximately 136 aminoacids (after cleavage of a 32 amino acid signal peptide) withdistinctive lysine and arginine-rich clusters within both N- andC-terminal domains. The complete cDNA sequence encoding PTN is providedin SEQ ID NO. 21, and the complete amino acid sequence of PTN isprovided in SEQ ID NO. 22. PTN is a 18 kDa, single chain, secretedprotein with 10 conserved disulfide linked cysteine residues.

[0185] As used herein, a compound that specifically binds to PTN is anycompound (such as an antibody) that has a binding affinity for anynaturally occurring isoform, splice variant, or polymorphism of PTN,explicitly including the isoforms described herein. As one of ordinaryskill in the art will appreciate, such “specific” binding compounds(e.g., antibodies) may also bind to other closely related proteins thatexhibit significant homology (such as greater than 90% identity, morepreferably greater than 95% identity, and most preferably greater than99% identity) with the amino acid sequence of PTN. Such proteins includetruncated forms or domains of PTN, and recombinantly engineeredalterations of PTN. For example, a portion of SEQ ID NO. 22 may beengineered to include a non-naturally occurring cysteine for crosslinking to an immunoconjugate protein, as described.

[0186] According to Milner et al. the gene sequence of PTN isolated fromhuman genomic DNA consists of five exons and four introns. While exon 1does not encode an amino acid sequence, exon 2 encodes the hydrophobicsignal sequence of 32 amino acids, exons 3 and 4 code for the aminoterminal and the ten cysteine residues, and exon 5 codes for the highlybasic C-terminal domains. Interestingly, the human cDNA starts towardthe end of exon 1, while the coded for protein begins at exon 2. Thus,the mature protein consist of 136 amino acids encoded by exons 2 to 5.As reported by Kretschmer et al. the minimum size of the gene is 42 kb,with a mRNA of 1650 nucleotides, spanning five exons, the majority ofthe protein being coded for by exon 3 (174 base pairs in length) andexon 4 (162 base pairs in length). See Kretschmer et al. (1993).Biochem. Biophys. Res. Commun. 192:420-429.

[0187] When raising antibodies to PTN, the entire protein or a portionthereof may be utilized. For instance, amino acid domains encoded for byexons 3 and 4 (i.e. amino acids 7 to 64 or 65 to 118, respectfully).Specifically, residues 41 to 64 may be used to abolish thetransformation potential of PTN. Larger PTN proteins and domains may beproduced utilizing any suitable recombinant vector/protein productionsystem, such as the baculovirus transfection system outlined below,after being amplified from a fetal brain cDNA library (as availablefrom, e.g., Clontech, Palo Alto, Calif.) or another suitable source. Itis to be noted that antibodies which bind to this secreted protein areuseful in cytotoxic and imaging embodiments of the invention, as one ofordinary skill would expect that the concentration of the PTN would beincreased adjacent to tumor cells which over-express the protein.

[0188] When utilizing an entire protein, or a larger section of theprotein, antibodies may be raised by immunizing the production animalwith the protein and a suitable adjuvant (e.g., Fruend's, Fruend'scomplete, oil-in-water emulsions, etc.). In these cases, the PTN protein(or a portion thereof) can serve as the PTN antigen. When a smallerpeptide is utilized, it is advantageous to conjugate the peptide with alarger molecule to make an immunostimulatory conjugate for use as thePTN antigen. Commonly utilized conjugate proteins that are commerciallyavailable for such use include bovine serum albumin (BSA) and keyholelimpet hemocyanin (KLH). In order to raise antibodies to particularepitopes, peptides derived from the full PTN sequence may be utilized.Preferably, one or more 8-30 aa peptide portions of the protein areutilized, with peptides in the range of 10-20 being a more economicalchoice. Custom-synthesized peptides in this range are available from amultitude of vendors, and can be order conjugated to KLH or BSA.Alternatively, peptides in excess of 30 amino acids may be synthesizedby solid-phase methods, or may be recombinantly produced in a suitablerecombinant protein production system. In order to ensure proper proteinglycosylation and processing, an animal cell system (e.g., Sf9 or otherinsect cells, CHO or other mammalian cells) is preferred. Otherinformation useful in designing an antigen for the production ofantibodies to PTN, including glycosylation sites, is provided in SEQ IDNO. 22.

[0189] PTN has been shown to bind to extracellular domain of RPTP betaand zeta. This binding inactivates the catalytic activity of RPTP, andPTN binds all the three major isoforms pf RPTP beta and zeta. PTN hasalso been shown to interact with syndecan-3. Thus, in alternativeembodiments of the compositions and methods of the invention, antibodymoieties are utilized which bind to PTN at a site on the protein thatalters the binding of a cell surface molecule, such as the ones listedabove, to PTN. Such PTN activity altering antibodies may be utilized intherapeutic compositions in an unconjugated form (e.g., the antibody inan acceptable pharmaceutical carrier), or may be conjugated to either atherapeutic moiety (creating a double-acting therapeutic agent) or animaging moiety (creating a duel therapeutic/imaging agent).

[0190] Selection of antibodies which alter (enhance or inhibit) thebinding of a ligand to PTN may be accomplished by a straightforwardbinding inhibition/enhancement assay. According to standard techniques,the binding of a labeled (e.g., fluorescently or enzyme-labeled)antibody to PTN, which has been immobilized in a microtiter well, isassayed in both the presence and absence of the appropriate ligand. Thechange in binding is indicative of either an enhancer (increasedbinding) or competitive inhibitor (decreased binding) relationshipbetween the antibody and the ligand. Such assays may be carried out inhigh-throughput formats (e.g., 384 well plate formats, in roboticsystems) for the automated selection of monoclonal antibody candidatesfor use as PTN ligand-binding inhibitors or enhancers.

[0191] In addition, antibodies which are useful for altering thefunction of PTN may be assayed in functional formats, such as the HUVECtube assay and the cell migration assay described below. Thus,antibodies that exhibit the appropriate anti-PTN activity may beselected without direct knowledge of a binding ligand or the particularbiomolecular interactions of PTN.

[0192] OPN

[0193] Given the experiments described above, and the results of Table1, OPN was selected as a target for selective immuno-therapeutic agentsin targeting and/or imaging brain tumors. The mature protein consists ofapproximately 298 amino acids (after cleavage of a 16 amino acid signalpeptide) and contains two potential Asn-Xaa-Ser N-glycosylation site,located at positions 65 and 92 of the mature protein. The complete cDNAsequence encoding OPN is provided in SEQ ID NO. 23, and the completeamino acid sequence of OPN is provided in SEQ ID NO. 24. OPN is anabundant 34 kDa, single chain, phosphorylated glycoprotein, with apresumed site for cell attachment at residues 144-148. Three isoformshave been identified to be generated by post transcriptionalmodification, such as alternative splicing, OPN-A, OPN-B, and OPN-C.OPN-A and OPN-B differ by the addition of 14 amino acids at residue 58of the protein. Amino acids 58-71 are absent in OPN-B, and amino acids31-57 are absent in OPN-C. OPN is a negatively charged, highlyhydrophilic secreted protein.

[0194] As used herein, a compound that specifically binds to OPN is anycompound (such as an antibody) that has a binding affinity for anynaturally occurring isoform, splice variant, or polymorphism of OPN,explicitly including the three isoforms described herein. As one ofordinary skill in the art will appreciate, such “specific” bindingcompounds (e.g., antibodies) may also bind to other closely relatedproteins that exhibit significant homology (such as greater than 90%identity, more preferably greater than 95% identity, and most preferablygreater than 99% identity) with the amino acid sequence of OPN. Suchproteins include truncated forms or domains of OPN, and recombinantlyengineered alterations of OPN. For example, a portion of SEQ ID NO. 24may be engineered to include a non-naturally occurring cysteine forcross linking to an immunoconjugate protein, as described.

[0195] According to Young et al. the cDNA sequence of OPN isolated fromhuman bone cells (OPN-A) has an overall structure of approximately 34kDA that consist of 298 amino acids, which is 14 amino acids less thanthe cDNA sequence of OPN isolated from human osteosarcoma by Keifer etal. (OPN-B). The cDNA transcript for OPN-A is 1.5 kb with an openreading frame of 900 nucleotides, of which the first 16 amino acids arehydrophobic in nature and probably constitute a signal sequence for thesecreted protein. The OPN gene contains 7 exons that are alternativelyspliced to generate the variant isoforms, the most common variant beingthe addition of a 42 bp (14 amino acid) sequence located at base 280 ofOPN-A. See Young et al. (1990). Genomics, 7:491-502 and Keifer et al.Nucleic Acids Res. 17:3306.

[0196] When raising antibodies to OPN, the entire protein or a portionthereof may be utilized. For instance, amino acid domains 4 to 12 (fromthe N-terminus) or 29 to 37 (from the N-terminus) may be utilized.Larger OPN proteins and domains may be produced utilizing any suitablerecombinant vector/protein production system, such as the baculovirustransfection system outlined below, after being amplified from a fetalbrain cDNA library (as available from, e.g., Clontech, Palo Alto,Calif.) or another suitable source. It is to be noted that antibodieswhich bind to this secreted protein are useful in cytotoxic and imagingembodiments of the invention, as one of ordinary skill would expect thatthe concentration of OPN would be increased adjacent to tumor cellswhich over-express the protein.

[0197] When utilizing an entire protein, or a larger section of theprotein, antibodies may be raised by immunizing the production animalwith the protein and a suitable adjuvant (e.g., Fruend's, Fruend'scomplete, oil-in-water emulsions, etc.). In these cases, the OPN protein(or a portion thereof) can serve as the OPN antigen. When a smallerpeptide is utilized, it is advantageous to conjugate the peptide with alarger molecule to make an immunostimulatory conjugate for use as theOPN antigen. Commonly utilized conjugate proteins that are commerciallyavailable for such use include bovine serum albumin (BSA) and keyholelimpet hemocyanin (KLH). In order to raise antibodies to particularepitopes, peptides derived from the full OPN sequence may be utilized.Preferably, one or more 8-30 aa peptide portions of the protein areutilized, with peptides in the range of 10-20 being a more economicalchoice. Custom-synthesized peptides in this range are available from amultitude of vendors, and can be order conjugated to KLH or BSA.Alternatively, peptides in excess of 30 amino acids may be synthesizedby solid-phase methods, or may be recombinantly produced in a suitablerecombinant protein production system. In order to ensure proper proteinglycosylation and processing, an animal cell system (e.g., Sf9 or otherinsect cells, CHO or other mammalian cells) is preferred. Otherinformation useful in designing an antigen for the production ofantibodies to OPN, including glycosylation sites, is provided in SEQ IDNO. 22.

[0198] The cell attachment sequence of human OPN (amino acids 144 to148) is believed to interact with various cell surface proteins (such asCD-44) to affect cell adhesion, and a highly acidic stretch composedalmost exclusively of aspartic acid residues (amino acids 72 to 81) isbelieved to be the mineral binding site within the protein. BecauseCD-44 is frequently over expressed on primary brain tumors andmetastases the binding of OPN to these various cell-surface adhesionprotein molecules may play a significant role in the senescence andgrowth of tumor cells in the brain. Thus, in alternative embodiments ofthe compositions and methods of the invention, antibody moieties areutilized which bind to OPN at a site on the protein that alters thebinding of a cell surface molecule, e.g., CD-44, to OPN. Such OPNactivity altering antibodies may be utilized in therapeutic compositionsin an unconjugated form (e.g., the antibody in an acceptablepharmaceutical carrier), or may be conjugated to either a therapeuticmoiety (creating a double-acting therapeutic agent) or an imaging moiety(creating a duel therapeutic/imaging agent).

[0199] Selection of antibodies which alter (enhance or inhibit) thebinding of a ligand to OPN may be accomplished by a straightforwardbinding inhibition/enhancement assay. According to standard techniques,the binding of a labeled (e.g., fluorescently or enzyme-labeled)antibody to OPN, which has been immobilized in a microtiter well, isassayed in both the presence and absence of the appropriate ligand. Thechange in binding is indicative of either an enhancer (increasedbinding) or competitive inhibitor (decreased binding) relationshipbetween the antibody and the ligand. Such assays may be carried out inhigh-throughput formats (e.g., 384 well plate formats, in roboticsystems) for the automated selection of monoclonal antibody candidatesfor use as OPN ligand-binding inhibitors or enhancers.

[0200] In addition, antibodies which are useful for altering thefunction of OPN may be assayed in functional formats, such as the HUVECtube assay and the cell migration assay described below. Thus,antibodies that exhibit the appropriate anti-OPN activity may beselected without direct knowledge of a the biomolecular role of OPN.

[0201] PTPζ

[0202] PTPζ was also selected as a prime target for selectiveimmuno-therapeutic agents in treating or imaging brain tumors. Thecomplete cDNA sequence encoding PTPζ is provided in SEQ ID NO. 5, andthe complete amino acid sequence of PTPζ is provided in SEQ ID NO. 6.Three different splice variants have been described, which include twomembrane bound variants (full length: PTPζ-α, and shorter versionPTPζ-β) and one secreted form (Phosphacan). See FIG. 1. Isoform PTPζ-αis the full length isoform, which contains the primary amino acidsequence aa 25-2314 of SEQ ID NO. 6 (aa 1-24 are a signal polypeptide).This full length long form of PTPζ is a type I membrane protein. Afterthe signal peptide it contains a carbonic anhydrase like (CAH) and afibronectin type III like (FN3) domain, followed by a long cysteine freespacer (S) domain. This follows a 860 amino acid long insert domain,which can be glycosylated. After a single transmembrane segment, in theintracellular region it has 2 phosphatase domains, but only themembrane-proximal PTPase domain is catalytically active (Krueger 1992).

[0203] In Isoform PTPζ-β, 755-1614 are missing. Isoform PTPζ-S(phosphacan), is a secreted isoform, which is comprises theextracellular domains of PTPζ-α. Northern Blot analysis have shown thatthe PTP zeta is exclusively expressed in the human central nervoussystem. In mouse embryos, the PTPζ transcript was mainly detected in theventricular and subventricular zone of the brain and the spinal cord.The same pattern was detected in adult mice. Detailed studies have shownthat during rat embryogenesis the two transmembrane splice variants ofPTPζ are mainly expressed in glial precursor cells and that thesecretory version (Phosphacan) is more abundant in mature astrocyteswhich have already migrated in the ventricle zone. Applicants havecharacterized two additional novel slice variants, PTPζ SM1 and PTPζSM2, which are described in detail below.

[0204] As used herein, a compound which specifically binds to humanprotein tyrosine phosphatase-zeta (PTPζ) is any compound (such as anantibody) which has a binding affinity for any naturally occurringisoform, spice variant, or polymorphism of PTPζ, explicitly includingthe three splice variants describe herein. For example, the compoundswhich specifically bind to novel isoforms PTPζ SM1 and PTPζ SM2,described below, are subsets of compounds which specifically bind toPTPζ. As one of ordinary skill in the art will appreciate, such“specific” binding compounds (e.g., antibodies) may also bind to otherclosely related proteins which exhibit significant homology (such asgreater than 90% identity, more preferably greater than 95% identity,and most preferably greater than 99% identity) with the amino acidsequence of PTPζ. Such proteins include truncated forms or domains ofPTPζ, and recombinantly engineered alterations of PTPζ. For example, anportion of SEQ ID NO. 6 may be engineered to include a non-naturallyoccurring cysteine for cross-linking to an immunoconjugate protein, asdescribed below.

[0205] In general, it is preferred that the antibodies utilized in thecompositions and methods of the invention bind to the membrane-boundisoforms of the protein, as this will more specifically target thecytotoxic therapeutic agent, or the imaging agent, to the brain tumorcell. However, embodiments which utilize antibodies which bind to thesecreted isoform of the protein are also useful in the invention, as oneof ordinary skill would expect that the concentration of the secretedisoform would also be increased adjacent to brain tumor cells whichover-express the protein.

[0206] The amino acid sequence of full length PTPζ consists of 2307amino acids, as the sequence was deduced by Levy (in which aa 1722-1728of SEQ ID NO. 2 were missing) (See also U.S. Pat. Nos. 5,604,094, and6,160,090, fully incorporated herein by reference), or 2314 amino acidsas the sequence was deduced by Krueger, et al., (“A human transmembraneprotein-tyrosine phosphatase, PTP zeta, is expressed in brain and has anN-terminal receptor domain homologous to carbonic anhydrases” Proc. Nat.Acad. Sci. U.S.A. 89:7417-7421 (1992)). Amino acids 1-24 of SEQ ID NO. 6are a signal sequence which directs the proper placement of thetransmembrane protein. The extracellular domain of the mature PTPζprotein spans amino acids 25-1635 of SEQ ID NO. 6 in the long andsecreted forms (this forms the entire secreted form), and amino acids25-754,1615-1635 in the short isoform. The transmembrane region of theprotein spans amino acids 1636-1661 of SEQ ID NO. 6, and the balance ofthe protein forms the cytoplasmic domain, amino acids 1662-2314.

[0207] When raising antibodies to PTPζ, the entire protein (any of thethree isoforms) or a portion thereof may be utilized. For instance, theextracellular domain of the long or short form, the entire secretedform, or a portion of extracellular domain may be utilized. Forinstance, amino acids 25-754, which are common to both α and β isoforms,may be used. Such larger PTPζ proteins and domains may be producedutilizing any suitable recombinant vector/protein production system,such as the baculovirus transfection system outlined below, after beingamplified from a fetal brain cDNA library (as available from, e.g.,Clontech, Palo Alto, Calif.) or another suitable source. When utilizingan entire protein, or a larger section of the protein, antibodies may beraised by immunizing the production animal with the protein and asuitable adjuvant (e.g., Fruend's, Fruend's complete, oil-in-wateremulsions, etc.). In these cases, the PTPζ protein (or a portionthereof) can serve as the PTPζ antigen. When a smaller peptide isutilized, it is advantageous to conjugate the peptide with a largermolecule to make an immunostimulatory conjugate for use as the PTPζantigen. Commonly utilized conjugate proteins which are commerciallyavailable for such use include bovine serum albumin (BSA) and keyholelimpet hemocyanin (KLH). In order to raise antibodies to particularepitopes, peptides derived from the full PTPζ sequence may be utilized.Preferably, one or more 8-30 aa peptide portions of an extracellulardomain of PTPζ are utilized, with peptides in the range of 10-20 being amore economical choice. Custom-synthesized peptides in this range areavailable from a multitude of vendors, and can be order conjugated toKLH or BSA. Alternatively, peptides in excess of 30 amino acids may besynthesized by solid-phase methods, or may be recombinantly produced ina suitable recombinant protein production system. In order to ensureproper protein glycosylation and processing, an animal cell system(e.g., Sf9 or other insect cells, CHO or other mammalian cells) ispreferred. Other information useful in designing an antigen for theproduction of antibodies to PTPζ, including glycosylation sites, isprovided in SEQ ID NO. 6.

[0208] The extracellular domain of human PTPζ is known to bind totenascin-C, tenascin-R, pleiotrophin (NM_(—)002825), midkine(NM_(—)002391), FGF-2 (XM_(—)00366), Nr-CAM 1NM_(—)005010), L1/Ng-CAM,contactin (NM_(—)001843), N-CAM (XM_(—)006332), andaxonin-1NM_(—)005076.) The first 5 of these molecules are eithercomponents of the extracellular matrix in gliomas or are soluble factorsknown to be present in gliomas, and the latter 4 are neuronal surfacemolecules. The binding of PTPζ to these molecules may play a significantrole in the oncogenesis and growth of neoplastic cells in the brain.Thus, in alternative embodiments of the compositions and methods of theinvention, antibody moieties are utilized which bind to PTPζ at a siteon the protein which alters the binding of an extracellular ligandmolecule to PTPζ. Such PTPζ activity altering antibodies may be utilizedin therapeutic compositions in an unconjugated form (e.g., the antibodyin an acceptable pharmaceutical carrier), or may be conjugated to eithera therapeutic moiety (creating a double-acting therapeutic agent) or animaging moiety (creating a duel therapeutic/imaging agent).

[0209] Selection of antibodies which alter (enhance or inhibit) thebinding of a ligand to PTPζ may be accomplished by a straightforwardbinding inhibition/enhancement assay. According to standard techniques,the binding of a labeled (e.g., fluorescently or enzyme-labeled)antibody to PTPζ, which has been immobilized in a microtiter well, isassayed in both the presence and absence of the ligand. The change inbinding is indicative of either an enhancer (increased binding) orcompetitive inhibitor (decreased binding) relationship between theantibody and the ligand. Such assays may be carried out inhigh-throughput formats (e.g., 384 well plate formats. in roboticsystems) for the automated selection of monoclonal antibody candidatesfor use as PTPζ ligand-binding inhibitors or enhancers.

[0210] In addition, antibodies which are useful for altering thefunction of PTPζ may be assayed in functional formats, such as the HUVECtube assay and the cell migration assay described below. Thus,antibodies that exhibit the appropriate anti-PTPζ activity may beselected without direct knowledge of a the biomolecular role of PTPζ.

[0211] Novel PTPζ Splice Variants PTPζ SM1 and PTPζ SM2

[0212] In addition to the known variants of PTPζ for use in theinvention, applicants have identified two novel splice variant isoformsof PTPζ, SM1 and SM2, from their clone libraries, see FIG. 2. Thesenovel isoforms, PTPζ SM1 and PTPζ SM2, differ in structure from thethree known isoforms heretofore disclosed, as is illustrated in FIG. 3.As only cDNA sequences for the known splice variants had been previouslydisclosed, rather than the full gene sequence, applicants verified thelocation of the novel sequences by comparison of the known splicevariant sequences and the novel sequences with a publicly availablegenomic sequence database.

[0213] The protein PTPζ SM1 (amino acid sequence SEQ ID NO. 2, cDNAsequence SEQ ID NO. 1) comprises the amino acids encoded by the firstnine exons of PTPζ-α, with three unique additional carboxy terminalamino acids, see FIG. 2. These are encoded by additional 3′ mRNAsequence (nucleotides 1262-1272 of SEQ ID NO. 1) from the intron of thegene between exons nine and ten. The PTPζ SM1 clone was isolated from ahuman fetal brain cDNA library, an has been shown to be expressed inseveral human glioblastoma cell lines. Expression of the SM1 splicevariant has also been confirmed in primary brain tumor samples. Theprotein comprises only extracellular domains of PTPζ, and is expected tobe secreted by the cell. Thus, PTPζ SM1 may serve a cell signaling ormessenger function, and may have bind to a receptor on the surface ofcells which are associated with or part of central nervous systemtissues. Thus, antibodies specific for PTPζ SM1, and not specific forthe other splicing isoforms of PTPζ, may be especially efficacious inthe brain tumor therapeutic or imaging compositions of the invention.The PTPζ SM1 protein mainly comprises the carbonic anhydrase-like domainwhich has been identified in PTPζ α.

[0214] Applicants have explored the relationship between the putativecarbonic anhydrase domain of PTPζ SM1 (SEQ ID NO. 2) and other humancarbonic anhydrase domains from carbonic anhydrase III (SEQ ID NO. 25),carbonic anhydrase I (SEQ ID NO. 26), and carbonic anhydrase VIX [e](SEQ ID NO. 27), shown below:1                                                   50          cah3human ˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜AKEW GYASHNGPDH          cah1human ˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜ASPDW GYDDKNGPEQ          cahehuman ˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜ML FSALLLEVIW ILAADGGQHW TYEGPHGQDHrptpzetaexon9_frame1 MRILKRFLAC IQLLCVCRLD WANGYYRQQR KLVEEIG..WSYTGALNQKN 51                                                 100          cah3_human WHELFPNAKG ENQSPIELHT KDIRHD...P SLQPWSVSYDGGSAKTILNN           cah1_human WSKLYPIANG NNQSPVDIKT SETKHD...TSLKPISVSYN PATAKEIINV           cahe_human WPASYPECGN NAQSPIDIQTDSVTFDPDLP ALQPHGYDQP GTEPLDLHNN rptpzetaexon9_frame1 WGKKYPTCNSPKQSPINIDE DLTQVNVNLK KLKFQGWDKT SLENTFIHNT101                                                150          cah3_human GKTCRVVFDD TYDRSMLRGG PLPGPYRLRQ FHLHWGS.S.DDHGSEHTVD           cah1_human GHSFHVNFED NDNRSVLKGG PFSDSYRLFQFHFHWGS.T. NEHGSEHTVD           cahe_human GHTVQLSLP. ....STLYLGGLPRKYVAAQ LHLHWGQ.KG SPGGSEHQIN rptpzetaexon9_frame1 GKTVEINLTNDYRVS...GG VSEMVFKASK ITFHWGKCNM SSDGSEHSLE151                                                200          cah3_human GVKYAAELHL VHWN.PKYNT FKEALKQRDG IAVIGIFLKIGH.ENGEFQI           cah1_human GVKYSAELHV AHWNSAKYSS LAEAASKADGLAVIGVLMKV GE.ANPKLQK           cahe_human SEATFAELHI VHYDSDSYDSLSEAAERPQG LAVLGILIEV GETKNIAYEH rptpzetaexon9_frame1 GQKFPLEMQIYCFDADRFSS FEEAVKGKGK LRALSILFEV GTEENLDFKA201                                                250          cah3_human FLDALDKIKT KGKEAPFTKF DPSCLFPACR .DYWTYQGSFTTPPCEECIV           cah1_human VLDALQAIKT KGKRAPFTNF DPSTLLPSSL.DFWTYPGSL THPPLYESVT           cahe_human ILSHLHEVRH KDQKTSVPPFNLRELLPKQL GQYFRYNGSL TTPPCYQSVL rptpzetaexon9_frame1 IIDGVESVSRFGKQAALDPF ILLNLLPNST DKYYIYNGSL TSPPCTDTVD251                                                300          cah3_human WLLLKEPMTV SSDQMAKLRS LLSSAENEPP VP...LVSNWRPPQPINNRV           cah1_human WIICKESISV SSEQLAQFRS LLSNVEGDNAVP...MQHNN RPTQPLKGRT           cahe_human WTVFYRRSQI SMEQLEKLQGTLFSTEEEPS KL...LVQNY RALQPLNQRM rptpzetaexon9_frame1 WIVFKDTVSISESQLAVFCE VLTMQQSGYV MLMDYLQNNF REQQYKFSRQ301                                                350          cah3_human VRASFK˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜          cah1_human VRASF˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜          cahe_human VFASFIQAGS SYTTGEMLSL GVGILVGCLC LLLAVYFIARKIRKKRLENR rptpzetaexon9_frame1 VFSSYTGKEE IHEAVCSSEP ENVQADPENYTSLLVTWERP RVVYDTMIEK 351                          380          cah3_human ˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜           cah1_human˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜           cahe_human KSVVFTSAQATTEA˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜ rptpzetaexon9_frame1 FAVLYQQLDG EDQTKHEFLTDGYQDLVTI*

[0215] Based on alignment with these catalytically active carbonicanhydrases, it seems unlikely that the CA domain could function as acarbonic anhydrase enzyme. Two of the three histidines implicated inbinding of the catalytic zinc are missing from the CA domain of thereceptor. In active enzymes there is a conserved HxHWG{18,20}ELH motif(the three histidines bind zinc), however, in the receptor this ismodified to TFHWG {18,20}EMQ; i.e. two of the three critical zinc atomswould be missing. For comparison, it has been found that a carbonicanhydrase related protein (CAH 8) that lacks just one of thesehistidines also lacks catalytic activity.

[0216] The protein PTPζ SM2 (amino acid sequence SEQ ID NO. 4) comprisesthe amino acids encoded by all exons of PTPζ-α, plus a 116 nucleotide“extra” exon, in the correct reading frame, between exons 23 and 24(nucleotides 6229-6345 of SEQ ID NO. 3). This extra exon, designatedexon 23a, contains a portion of the intron sequence between exons 23 and24 of the PTPζ gene. PTPζ SM2 expression has been verified in severalhuman glioblastoma cell lines, and has also been confirmed in primarybrain tumor samples. As PTPζ SM2 comprises all the domains of PTPζ α,the protein is expected to be membrane-bound. The extra exon lies withinthe cytoplasmic domain of the protein, and thus may alter the proteintyrosine phosphatase function of PTPζ SM2.

[0217] A novel splicing variant PTPζ protein having an amino acidsequence which includes the amino acid sequence of PTPζ SM1 (SEQ ID NO.2) or PTPζ SM2 (SEQ. ID NO. 4) may be produced by recombinant techniquesknown in the art utilizing any suitable vector, in any suitable hostcell. The term “vector” is intended to include any physical orbiochemical vehicle containing nucleic acid polymers of interest, bywhich those nucleic acid polymers are transferred into a host cell,thereby transfecting that cell with the introduced nucleic acidpolymers. The transfected nucleic acid sequence preferably contains acontrol sequence, such as a promoter sequence, suitable fortranscription of the nucleic acid sequence in the host cell. Examples ofvectors include DNA plasmids, viruses, liposomes, particle gun pellets,and transfection vectors known to those of skill in the molecularbiology arts. The term “host cell” is intended to mean the target cellfor vector transformation, in which the transferred nucleic acid polymerwill be replicated and/or expressed. Although bacterial cells may besuitable for production of the proteins for antibody production orstructural study purposes, eukaryotic cell hosts are preferred forproduction of the protein for functional assays or therapeutic purposes.Preferred eukaryotic cell hosts include insect cell lines (e.g., Sf9,Sf21, or High Five™ cell lines), and mammalian cell lines (e.g., HeLa,CHO-K1, COS-7, COS-1, HEK293, HEPG2, Jurkat, MDCK, PAE, PC-12, and otheracceptable mammalian cell lines). Thus, the invention also providesvectors incorporating a nucleic acid sequence encoding PTPζ SM1 or PTPζSM2, as well as host cells which express the proteins.

[0218] It is common in the molecular biology arts to utilize additionalfunctional amino acid domains or proteins fused with a protein sequenceof interest for purification or detection purposes. Such additionalfunctionalities include, for example, polyhistidine domains, c-mycdomains (specifically comprising amino acids 410-419 of the human c-myconcogene product), β-galactosidase, β-glucuronidase,glutathione-S-transferase, maltose binding protein, human influenzavirus hemagglutanin, green fluorescent protein, chloramphenicolacetyltransferase, luciferase, thioredoxin, and others. Afterpurification (e.g., by antibody-affinity chromatography) or detection,these extra amino acid sequences may be cleaved (e.g., by thrombin,enterokinase, Factor Xa, or other protease) to yield a functional matureprotein. Thus, the PTPζ SM1 and SM2 proteins of the invention alsoencompass proteins comprising the amino acid sequence of SEQ ID NO. 2 orSEQ ID NO. 4 and such additional amino acid functionalities.

[0219] The invention also provides polypeptides which have a uniqueactivity of PTPζ SM1 or PTPζ SM2 which is not shared by the other PTPζsplice variant (e.g., an antigenic epitope) and which include a portionof the amino acid sequence of PTPζ SM1 or PTPζ SM2 which is at leastabout 8 to 12 amino acid residues in length, more preferably at leastabout 20 amino acids in length. These polypeptides preferably comprisean amino acid sequence which is not found in PTPζ α, PTPζ β, orphosphacan, wherein the included portion of the sequence confers theunique activity on the polypeptide. Such polypeptides may be utilized asdescribed above to produce affinity reagents which specifically bind toPTPζ splice variants SM1 or SM2, but do not bind to the other knownsplice variants of PTPζ. The invention thus provides such specificaffinity reagents, which may be produces from such polypeptides, or froman entire PTPζ SM1or PTPζ SM2 protein. In preferred embodiments theseaffinity reagents are antibodies or antibody fragments.

[0220] In addition, although the understanding of the field of proteinbiochemistry is not as complete as that of molecular genetics, theperson or ordinary skill in the art of biochemistry is capable ofpredicting, with reasonable certainty, when certain substitutions to theprimary amino acid sequence structure of a protein will not result inany appreciable modification of a protein's structure or function. Suchconservative substitutions are made by replacing an amino acid in thesequence with another containing a side chain with like charge, size,and other characteristics. Conservative substitutions in a proteinsequence which would be expected to have minimal to no impact on proteinstructure or function can be readily devised by a person of ordinaryskill in the biochemical arts. To the extent that such conservativesubstitutions can be made while retaining 90%, preferably 95%, and morepreferably 99% or more identity to SEQ. ID NO. 2 or SEQ ID NO. 4, andmaintain the activity of the native PTPζ SM1 or PTPζ SM2 protein, suchaltered proteins are within the scope of the present invention.

[0221] The invention also provides nucleic acid polymers encoding thePTPζ splice variants SM1 or SM2. These nucleic acid polymers mostpreferably comprises a nucleic acid sequence of SEQ. ID NO. 1 or SEQ IDNO. 3, or the predictable variants thereof which one of ordinary skillof the art could derive using the degeneracy of the genetic code. Suchnucleic acid polymers are useful for the production of PTPζ SM1 or PTPζSM2 by recombinant methods, as described above.

[0222] The invention also encompasses nucleic acid probes or primerswhich hybridize to the mRNA encoding PTPζ splice variants SM1 or SM2,but not mRNA encoding other known splice variants of PTPζ. Such probesor primers provided by the invention are preferably able to hybridizewith SEQ. ID NO. 1 or SEQ. ID NO. 3 (or their complements) understringent conditions (e.g., 0.5× to 2×SSC buffer, 0.1% SDS, and atemperature of 55-65° C.), but do not hybridize to SEQ ID NO. 5 (or itscomplement) under the same conditions. These PTPζ SM1 or PTPζ SM2 codingsequence specific probes are preferably from about 16 to about 40nucleotides in length, more preferably from about 18 nucleotides toabout 30 nucleotides in length. However, probes may be of a smallersize, preferably from about 8 to about 15 nucleotides in length, if twoore more probes are hybridized to adjacent sequences, so that terminalnucleic acid base-stacking interactions may stabilize theirhybridization. In preferred embodiments of PTPζ SM1 specific nucleicacid probes, the probes hybridize at or near the novel splice site atthe 3′ end of exon 9, or its complement. In preferred embodiments ofPTPζ SM2 specific probes, the probes hybridize at or adjacent to alocation selected from: the novel splice site at the 3′ end of exon 23,at least a portion of the novel exon 23a, the novel splice site at the5′ end of exon 24, or the complement of any one of these.

[0223] Because PTPζ SM1 and PTPζ SM2 have been shown to be expressed inglioblastoma cell lines and primary tumors, the level of the expressionof these splice variants may be useful for staging or characterizingglioblastoma cells. Such cells may be extracted, for instance, from aprimary tumor. Thus, the invention provides for the monitoring of therelative expression level of PTPζ SM1 or PTPζ SM2, or both, in relationto each other or to one or more of the known PTPζ splice variants. Inone preferred embodiment, the level of expression of PTPζ SM1 is compareto at least one other splice variant selected from PTPζ SM2, PTPζ α,PTPζ β, and phosphacan. In another preferred embodiment, the level ofexpression of PTPζ SM2 is compare to at least one other splice variantselected from PTPζ SM1, PTPζ α, PTPζ β, and phosphacan. Such comparisonmay be made in either a qualitative or quantitative manner. One meansfor comparison is by hybridizing splice-variant specific nucleic acidprobes to a sample of nucleic acids (which may be amplified) obtainedfrom brain tumor cells. Alternatively, the expression level of thesplice variants may be deduced by the amplification of splice variantnucleic acid sequences, and the analysis of the size of those amplifiedproducts using methods known in the art. In another alternativeembodiment, protein levels may be studied utilizing splice-variantspecific antibodies in either sandwich immunoassay or in-situ stainingformats. Various expression level assay techniques are known to those ofskill in the molecular biological arts, and thus the specific techniquesmentioned above should be considered merely exemplary.

[0224] Antibodies for Use in the Antibody-Therapeutics Methods of theInvention

[0225] Generally, as the term is utilized in the specification,“antibody” or “antibody moiety” is intended to include any polypeptidechain-containing molecular structure that has a specific shape whichfits to and recognizes an epitope, where one or more non-covalentbinding interactions stabilize the complex between the molecularstructure and the epitope. Antibodies which bind specifically to one ofthe brain tumor protein targets are referred to as anti-brain tumorprotein target antibodies, or α(T_(BT)), or more specifically α(ARP2),α(SPARC), α(CMET), α(CD44), α(BEHAB), α(TSPAN3), α(VIPR2), α(OPN),α(PTN), and α(PTPζ). The specific or selective fit of a given structureand its specific epitope is sometimes referred to as a “lock and key”fit. The archetypal antibody molecule is the immunoglobulin, and alltypes of immunoglobulins (IgG, IgM, IgA, IgE, IgD, etc.), from allsources (e.g., human, rodent, rabbit, cow, sheep, pig, dog, othermammal, chicken, turkey, emu, other avians, etc.) are considered to be“antibodies.” Antibodies utilized in the present invention may bepolyclonal antibodies, although monoclonal antibodies are preferredbecause they may be reproduced by cell culture or recombinantly, and maybe modified to reduce their antigenicity.

[0226] Polyclonal antibodies may be raised by a standard protocol byinjecting a production animal with an antigenic composition, formulatedas described above. See, e.g., Harlow and Lane, Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory, 1988. In one such technique, anT_(BT) antigen comprising an antigenic portion of the brain tumorprotein targets' polypeptide is initially injected into any of a widevariety of mammals (e.g., mice, rats, rabbits, sheep or goats).Alternatively, in order to generate antibodies to relatively shortpeptide portions of the brain tumor protein target (see discussionabove), a superior immune response may be elicited if the polypeptide isjoined to a carrier protein, such as ovalbumin, BSA or KLH. Thepeptide-conjugate is injected into the animal host, preferably accordingto a predetermined schedule incorporating one or more boosterimmunizations, and the animals are bled periodically. Polyclonalantibodies specific for the polypeptide may then be purified from suchantisera by, for example, affinity chromatography using the polypeptidecoupled to a suitable solid support.

[0227] Alternatively, for monoclonal antibodies, hybridomas may beformed by isolating the stimulated immune cells, such as those from thespleen of the inoculated animal. These cells are then fused toimmortalized cells, such as myeloma cells or transformed cells, whichare capable of replicating indefinitely in cell culture, therebyproducing an immortal, immunoglobulin-secreting cell line. The immortalcell line utilized is preferably selected to be deficient in enzymesnecessary for the utilization of certain nutrients. Many such cell lines(such as myelomas) are known to those skilled in the art, and include,for example: thymidine kinase (TK) or hypoxanthine-guaninephosphoriboxyl transferase (HGPRT). These deficiencies allow selectionfor fused cells according to their ability to grow on, for example,hypoxanthine aminopterinthymidine medium (HAT).

[0228] Preferably, the immortal fusion partners utilized are derivedfrom a line that does not secrete immunoglobulin. The resulting fusedcells, or hybridomas, are cultured under conditions that allow for thesurvival of fused, but not unfused, cells and the resulting coloniesscreened for the production of the desired monoclonal antibodies.Colonies producing such antibodies are cloned, expanded, and grown so asto produce large quantities of antibody, see Kohler and Milstein, 1975Nature 256:495 (the disclosures of which are hereby incorporated byreference).

[0229] Large quantities of monoclonal antibodies from the secretinghybridomas may then be produced by injecting the clones into theperitoneal cavity of mice and harvesting the ascites fluid therefrom.The mice, preferably primed with pristine, or some other tumor-promoter,and immunosuppressed chemically or by irradiation, may be any of varioussuitable strains known to those in the art. The ascites fluid isharvested from the mice and the monoclonal antibody purified therefrom,for example, by CM Sepharose column or other chromatographic means.Alternatively, the hybridomas may be cultured in vitro or as suspensioncultures. Batch, continuous culture, or other suitable culture processesmay be utilized. Monoclonal antibodies are then recovered from theculture medium or supernatant.

[0230] Several monoclonal antibodies against various isoforms of thebrain tumor protein targets are currently available from commercialsources. For instance, a non-exclusive list of available commercialantibodies includes: for SPARC/Osteonectin, from Zymed, mouseanti-bovine MAb (cross-reactivity with human), suitable for ELISA, WB,IH (paraffin), Cat# 33-5500; for c-MET, from Zymed, rabbit anti-humanpolyclonal, suitable for ELISA, WB, IH. Cat# 71-8000, and from RDI,rabbit anti-human MAb, suitable for WB, IP, IH. Cat# RDI-MET Cabr.; forCD44, from RDI, mouse anti-human MAb, only for IH and FACS, Cat#RD1-M1676clb., and from Lab vision, mouse anti-human MAb, known to blockbinding of hyaluronic acid to its receptor CD44, “CD44/H-CAM Ab-2”; forBrevican/BEHAB, from BD Transduction Lab., a mouse anti-human MAb, WB,IF, Cat# B68820; for VIP 2 receptor, from Exalpha, mouse anti-rat(possible human cross-specificity, which is easily assayed) MAb, WB, IH.Cat#2140M; for Laminin receptor 67 kDa, from Lab vision, mouseanti-human MAb, IH, ELISA, not for WB. “laminin receptor Ab-1”; forOsteopontin, from Chemicon, rat anti-human MAb, raised againstrh-Osteopontin—recognizes native protein well, WB, IH, ELISA. “MAB3057”;for Pleiotrophin, from R&D goat anti-human polyclonal, WB, recognizesrh-Pleiotrophin. “BAF252”, and from Oncogene goat anti-human polyclonal,WB, ELISA, detects rh-Pleiotrophin. “PC87L”.; for PTPζ-α and PTPζ-β,from BD Transduction Labs, mouse anti-human MAb (WB, IH, IF),denominated “R20720” and from Chemicon, mouse anti-human MAb (WB, IH,IP), denominated “MAB5210”, which recognizes both of the transmembraneisoforms, and also recognizes the soluble isoform (phosphacan, PTPζ-S).These antibodies are suitable for use in the compositions of the presentinvention, especially in Fab fragment form (which eliminates significantportions of the antigenic mouse constant heavy and light chain regions).However, it is preferred that such antibodies be humanized or chimerizedaccording to one of the procedures outlined below.

[0231] In addition, the antibodies or antigen binding fragments may beproduced by genetic engineering. In this technique, as with the standardhybridoma procedure, antibody-producing cells are sensitized to thedesired antigen or immunogen. The messenger RNA isolated from the immunespleen cells or hybridomas is used as a template to make cDNA using PCRamplification. A library of vectors, each containing one heavy chaingene and one light chain gene retaining the initial antigen specificity,is produced by insertion of appropriate sections of the amplifiedimmunoglobulin cDNA into the expression vectors. A combinatorial libraryis constructed by combining the heavy chain gene library with the lightchain gene library. This results in a library of clones which co-expressa heavy and light chain (resembling the Fab fragment or antigen bindingfragment of an antibody molecule). The vectors that carry these genesare co-transfected into a host (e.g. bacteria, insect cells, mammaliancells, or other suitable protein production host cell.). When antibodygene synthesis is induced in the transfected host, the heavy and lightchain proteins self-assemble to produce active antibodies that can bedetected by screening with the antigen or immunogen.

[0232] Preferably, recombinant antibodies are produced in a recombinantprotein production system which correctly glycosylates and processes theimmunoglobulin chains, such as insect or mammalian cells. An advantageto using insect cells which utilize recombinant baculoviruses for theproduction of antibodies for use in the present invention is that thebaculovirus system allows production of mutant antibodies much morerapidly than stably transfected mammalian cell lines. In addition,insect cells have been shown to correctly process and glycosylateeukaryotic proteins, which prokaryotic cells do not. Finally, thebaculovirus expression of foreign protein has been shown to constituteas much as 50-75% of the total cellular protein late in viral infection,making this system an excellent means of producing milligram quantitiesof the recombinant antibodies.

[0233] The use of the baculovirus Autographia californica nuclearpolyhedrosis virus (AcNPV) and recombinant viral stocks in Spodopterafrugiperda (Sf9) cells to prepare large quantities of protein has beendescribed by Smith et al. (1985), Summers and Smith (1987). A preferredmethod of preparing recombinant antibodies is through the expression ofDNA encoding recombinant antibody (produced by screening, as above, orby protein engineering to include more human-like domains, as discussedbelow) via the baculoviral expression system in Sf9 insect cells.Production of recombinant proteins in Sf9 cells is well known in theart, and one of ordinary skill would be able to select from a number ofacceptable protocols (e.g., that described in U.S. Pat. No. 6,603,905).

[0234] It should be noted that antibodies which have a reducedpropensity to induce a violent or detrimental immune response in humans(such as anaphylactic shock), and which also exhibit a reducedpropensity for priming an immune response which would prevent repeateddosage with the antibody therapeutic or imaging agent (e.g., thehuman-anti-murine-antibody “HAMA” response), are preferred for use inthe invention. These antibodies are preferred for all administrativeroutes, including intrathecal administration. Even through the brain isrelatively isolated in the cranial cavity, behind the blood brainbarrier, an immune response still can occur in the form of increasedleukocyte infiltration, and inflammation. Although some increased immuneresponse against the tumor is desirable, the concurrent binding andinactivation of the therapeutic or imaging agent generally outweighsthis benefit. Thus, humanized, chimeric, or xenogenic human antibodies,which produce less of an immune response when administered to humans,are preferred for use in the present invention.

[0235] Chimeric antibodies may be made by recombinant means by combiningthe murine variable light and heavy chain regions (VK and VH), obtainedfrom a murine (or other animal-derived) hybridoma clone, with the humanconstant light and heavy chain regions, in order to produce an antibodywith predominantly human domains. The production of such chimericantibodies is well known in the art, and may be achieved by standardmeans (as described, e.g., in U.S. Pat. No. 5,624,659, incorporatedfully herein by reference). Humanized antibodies are engineered tocontain even more human-like immunoglobulin domains, and incorporateonly the complementarity-determining regions of the animal-derivedantibody. This is accomplished by carefully examining the sequence ofthe hyper-variable loops of the variable regions of the monoclonalantibody, and fitting them to the structure of the human antibodychains. Although facially complex, the process is straightforward inpractice. See, e.g., U.S. Pat. No. 6,187,287, incorporated fully hereinby reference.

[0236] Alternatively, polyclonal or monoclonal antibodies may beproduced from animals which have been genetically altered to producehuman immunoglobulins, such as the Abgenix XenoMouse or the MedarexHuMAb® technology. The transgenic animal may be produced by initiallyproducing a “knock-out” animal which does not produce the animal'snatural antibodies, and stably transforming the animal with a humanantibody locus (e.g., by the use of a human artificial chromosome). Onlyhuman antibodies are then made by the animal. Techniques for generatingsuch animals, and deriving antibodies therefrom, are described in U.S.Pat. Nos. 6,162,963 and 6,150,584, incorporated fully herein byreference. Such fully human xenogenic antibodies are a preferredantibody for use in the methods and compositions of the presentinvention.

[0237] Alternatively, single chain antibodies (Fv, as described below)can be produced from phage libraries containing human variable regions.See U.S. Pat. No. 6,174,708, incorporated fully herein by reference.Also see Kuan, C. T., Reist, C. J., Foulon, C. F., Lorimer, I. A.,Archer, G., Pegram, C. N., Pastan, I., Zalutsky, M. R., and Bigner, D.D. (1999). 125I-labeled anti-epidermal growth factor receptor-viiisingle-chain Fv exhibits specific and high-level targeting of gliomaxenografts. Clin Cancer Res. 5, 1539-49;Lorimer, I. A.,Keppler-Hafkemeyer, A., Beers, R. A., Pegram, C. N., Bigner, D. D., andPastan, I. (1996). Recombinant immunotoxins specific for a mutantepidermal growth factor receptor: targeting with a single chain antibodyvariable domain isolated by phage display. Proc. Nat. Acad. Sci. USA 93,14815-20; Pastan, I. H., Archer, G. E., McLendon, R. E., Friedman, H.S., Fuchs, H. E., Wang, Q. C., Pai, L. H., Herndon, J., and Bigner, D.D. (1995). Intrathecal administration of single-chain immunotoxin, LMB-7[B3(Fv)-PE38], produces cures of carcinomatous meningitis in a ratmodel. Proc Natl. Acad. Sci USA 92, 2765-9, all of which areincorporated by reference fully herein.

[0238] In addition to entire immunoglobulins (or their recombinantcounterparts), immunoglobulin fragments comprising the epitope bindingsite (e.g., Fab′, F(ab′)₂, or other fragments) are useful as antibodymoieties in the present invention. Such antibody fragments may begenerated from whole immunoglobulins by ficin, pepsin, papain, or otherprotease cleavage. “Fragment,” or minimal immunoglobulins may bedesigned utilizing recombinant immunoglobulin techniques. For instance“Fv” immunoglobulins for use in the present invention may be produced bylinking a variable light chain region to a variable heavy chain regionvia a peptide linker (e.g., poly-glycine or another sequence which doesnot form an alpha helix or beta sheet motif).

[0239] Fv fragments are heterodimers of the variable heavy chain domain(V_(H)) and the variable light chain domain (V_(L)). The heterodimers ofheavy and light chain domains that occur in whole IgG, for example, areconnected by a disulfide bond. Recombinant Fvs in which V_(H) and V_(L)are connected by a peptide linker are typically stable, see, forexample, Huston et al., Proc. Natl. Acad, Sci. USA 85:5879-5883 (1988)and Bird et al., Science 242:423-426 (1988), both fully incorporatedherein, by reference. These are single chain Fvs which have been foundto retain specificity and affinity and have been shown to be useful forimaging tumors and to make recombinant immunotoxins for tumor therapy.However, researchers have bound that some of the single chain Fvs have areduced affinity for antigen and the peptide linker can interfere withbinding. Improved Fv's have been also been made which comprisestabilizing disulfide bonds between the V_(H) and V_(L) regions, asdescribed in U.S. Pat. No. 6,147,203, incorporated fully herein byreference. Any of these minimal antibodies may be utilized in thepresent invention, and those which are humanized to avoid HAMA reactionsare preferred for use in embodiments of the invention.

[0240] In addition, derivatized immunoglobulins with added chemicallinkers, detectable moieties [fluorescent dyes, enzymes, substrates,chemiluminescent moieties], or specific binding moieties [such asstreptavidin, avidin, or biotin] may be utilized in the methods andcompositions of the present invention. For convenience, the term“antibody” or “antibody moiety” will be used throughout to generallyrefer to molecules which specifically bind to an epitope of the braintumor protein targets, although the term will encompass allimmunoglobulins, derivatives, fragments, recombinant or engineeredimmunoglobulins, and modified immunoglobulins, as described above.

[0241] Candidate anti-T_(BT) antibodies can be tested for anti-T_(BT)activity by any suitable standard means. As a first screen, theantibodies may be tested for binding against the brain tumor proteintarget antigen utilized to produce them, or against the entire braintumor protein target extracellular domain or protein. As a secondscreen, anti-T_(BT) candidates may be tested for binding to anappropriate glioblastoma cell line (i.e., one which approximates primarytumor brain tumor protein target expression), or to primary tumor tissuesamples. For these screens, the anti-T_(BT) candidate antibody may belabeled for detection (e.g., with fluorescein or another fluorescentmoiety, or with an enzyme such as horseradish peroxidase). Afterselective binding to the brain tumor protein target is established, thecandidate antibody, or an antibody conjugate produced as describedbelow, may be tested for appropriate activity (i.e., the ability todecrease tumor cell growth and/or to aid in visualizing tumor cells) inan in vivo model, such as an appropriate glioblastoma cell line, or in amouse or rat human brain tumor model, as described below.

[0242] General Functional Assay Methods for Antibodies for Use in theInvention

[0243] In addition to the specific binding assays and protein-specificfunctional assays described for individual proteins above, antibodieswhich are useful for altering the function of ARP-2, SPARC, c-MET,BEHAB, CD-44, TSPN3, PTN, OPN, VIPR-2, or PTPζ may be assayed infunctional formats, such as glioblastoma cell culture or mouse/rat CNStumor model studies. In glioblastoma cell models of activity, expressionof the protein is first verified in the particular cell strain to beused. If necessary, the cell line may be stably transfected with acoding sequence of the protein under the control of an appropriateconstituent promoter, in order to express the protein at a levelcomparable to that found in primary tumors. The ability of theglioblastoma cells to survive in the presence of the candidatefunction-altering anti-protein antibody is then determined. In additionto cell-survival assays, cell migration assays, as described below inExample 1, may be utilized to determine the effect of the candidateantibody therapeutic agent on the tumor-like behavior of the cells.Alternatively, if the brain tumor protein target is involved inangiogenesis, or endothelial cell sprouting assays such as described inExample 2 may be utilized to determine the ability of the candidateantibody therapeutic to inhibit vascular neogenesis, an importantfunction in tumor biology.

[0244] Similarly, in vivo models for human brain tumors, particularlynude mice/SCID mice model or rat models, have been described [Antunes,L., Angioi-Duprez, K. S., Bracard, S. R., Klein-Monhoven, N. A., LeFaou, A. E., Duprez, A. M., and Plenat, F. M. (2000). Analysis of tissuechimerism in nude mouse brain and abdominal xenograft models of humanglioblastoma multiforme: what does it tell us about the models and aboutglioblastoma biology and therapy? J Histochem Cytochem 48, 847-58;Price, A., Shi, Q., Morris, D., Wilcox, M. E., Brasher, P. M.,Rewcastle, N. B., Shalinsky, D., Zou, H., Appelt, K., Johnston, R. N.,Yong, V. W., Edwards, D., and Forsyth, P. (1999). Marked inhibition oftumor growth in a malignant glioma tumor model by a novel syntheticmatrix metalloproteinase inhibitor AG3340. Clin Cancer Res 5, 845-54;and Senner, V., Sturm, A., Hoess, N., Wassmann, H., and Paulus, W.(2000). In vivo glioma model enabling regulated gene expression. ActaNeuropathol (Berl) 99, 603-8.] Once correct expression of the protein inthe tumor model is verified, the effect of the candidate anti-proteinantibodies on the tumor masses in these models can be evaluated, whereinthe ability of the anti-protein antibody candidates to alter proteinactivity is indicated by a decrease in tumor growth or a reduction inthe tumor mass. Thus, antibodies that exhibit the appropriate anti-tumoreffect may be selected without direct knowledge of the particularbiomolecular role of the protein in oncogenesis.

[0245] Therapeutic and Imaging Moieties and Methods for Conjugating ThemQith anti-PTPζ Antibodies to Use in the Compositions and Methods of theInvention

[0246] As described above, the anti-T_(BT) antibodies for use in thepresent invention may have utility without conjugation when the nativeactivity of the brain tumor protein target is altered in the tumor cell.Such antibodies, which may be selected as described above, may beutilized without further modification to include a cytotoxic or imagingmoiety. These types of compositions have the advantage of reducedtoxicity (in that only the toxicity of the antibody moieties themselvesmust be taken into account when dosing), and are simpler to manufacture.Thus, non-conjugated activity altering anti-T_(BT) antibody therapeuticsare a preferred embodiment of the invention. However, the conjugation ofcytotoxic or imaging agents is yet another preferred embodiment whenutilizing these antibodies because the added moieties add functionalityto the therapeutic.

[0247] Thus, in many preferred embodiments of the invention, theanti-T_(BT) antibodies may be coupled or conjugated to one or moretherapeutic cytotoxic or imaging moieties. As used herein, “cytotoxicmoiety” (C) simply means a moiety which inhibits cell growth or promotescell death when proximate to or absorbed by the cell. Suitable cytotoxicmoieties in this regard include radioactive isotopes (radionuclides),chemotoxic agents such as differentiation inducers and small chemotoxicdrugs, toxin proteins, and derivatives thereof. As utilized herein,“imaging moiety” (I) means a moiety which can be utilized to increasecontrast between a tumor and the surrounding healthy tissue in avisualization technique (e.g., radiography, positron-emissiontomography, magnetic resonance imaging, direct or indirect visualinspection). Thus, suitable imaging moieties include radiographymoieties (e.g. heavy metals and radiation emitting moieties), positronemitting moieties, magnetic resonance contrast moieties, and opticallyvisible moieties (e.g., fluorescent or visible-spectrum dyes, visibleparticles, etc.). It will be appreciated by one of ordinary skill thatsome overlap exists between what is a therapeutic moiety and what is animaging moiety. For instance ²¹²Pb and ²¹²Bi are both usefulradioisotopes for therapeutic compositions, but are also electron-dense,and thus provide contrast for X-ray radiographic imaging techniques, andcan also be utilized in scintillation imaging techniques.

[0248] In general, therapeutic or imaging agents may be conjugated tothe anti-PTPζ moiety by any suitable technique, with appropriateconsideration of the need for pharmokinetic stability and reducedoverall toxicity to the patient. A therapeutic agent may be coupled to asuitable antibody moiety either directly or indirectly (e.g. via alinker group). A direct reaction between an agent and an antibody ispossible when each possesses a functional group capable of reacting withthe other. For example, a nucleophilic group, such as an amino orsulfhydryl group, may be capable of reacting with a carbonyl-containinggroup, such as an anhydride or an acid halide, or with an alkyl groupcontaining a good leaving group (e.g., a halide). Alternatively, asuitable chemical linker group may be used. A linker group can functionas a spacer to distance an antibody from an agent in order to avoidinterference with binding capabilities. A linker group can also serve toincrease the chemical reactivity of a substituent on a moiety or anantibody, and thus increase the coupling efficiency. An increase inchemical reactivity may also facilitate the use of moieties, orfunctional groups on moieties, which otherwise would not be possible.

[0249] Suitable linkage chemistries include maleimidyl linkers and alkylhalide linkers (which react with a sulfhydryl on the antibody moiety)and succinimidyl linkers (which react with a primary amine on theantibody moiety). Several primary amine and sulfhydryl groups arepresent on immunoglobulins, and additional groups may be designed intorecombinant immunoglobulin molecules. It will be evident to thoseskilled in the art that a variety of bifunctional or polyfunctionalreagents, both homo- and hetero-functional (such as those described inthe catalog of the Pierce Chemical Co., Rockford, Ill.), may be employedas a linker group. Coupling may be effected, for example, through aminogroups, carboxyl groups, sulfhydryl groups or oxidized carbohydrateresidues. There are numerous references describing such methodology,e.g., U.S. Pat. No. 4,671,958. As an alternative coupling method,cytotoxic or imaging moieties may be coupled to the anti-T_(BT) antibodymoiety through a an oxidized carbohydrate group at a glycosylation site,as described in U.S. Pat. Nos. 5,057,313 and 5,156,840. Yet anotheralternative method of coupling the antibody moiety to the cytotoxic orimaging moiety is by the use of a non-covalent binding pair, such asstreptavidin/biotin, or avidin/biotin. In these embodiments, one memberof the pair is covalently coupled to the antibody moiety and the othermember of the binding pair is covalently coupled to the cytotoxic orimaging moiety.

[0250] Where a cytotoxic moiety is more potent when free from theantibody portion of the immunoconjugates of the present invention, itmay be desirable to use a linker group which is cleavable during or uponinternalization into a cell, or which is gradually cleavable over timein the extracellular environment. A number of different cleavable linkergroups have been described. The mechanisms for the intracellular releaseof a cytotoxic moiety agent from these linker groups include cleavage byreduction of a disulfide bond (e.g., U.S. Pat. No. 4,489,710), byirradiation of a photolabile bond (e.g., U.S. Pat. No. 4,625,014), byhydrolysis of derivatized amino acid side chains (e.g., U.S. Pat. No.4,638,045), by serum complement-mediated hydrolysis (e.g., U.S. Pat. No.4,671,958), and acid-catalyzed hydrolysis (e.g., U.S. Pat. No.4,569,789).

[0251] It may be desirable to couple more than one cytotoxic and/orimaging moiety to an antibody. By poly-derivatizing the anti-T_(BT)antibody, several cytotoxic strategies may be simultaneouslyimplemented, an antibody may be made useful as a contrasting agent forseveral visualization techniques, or a therapeutic antibody may belabeled for tracking by a visualization technique. In one embodiment,multiple molecules of an imaging or cytotoxic moiety are coupled to oneantibody molecule. In another embodiment, more than one type of moietymay be coupled to one antibody. Regardless of the particular embodiment,immunoconjugates with more than one moiety may be prepared in a varietyof ways. For example, more than one moiety may be coupled directly to anantibody molecule, or linkers which provide multiple sites forattachment (e.g., dendrimers) can be used. Alternatively, a carrier withthe capacity to hold more than one cytotoxic or imaging moiety can beused.

[0252] A carrier may bear the agents in a variety of ways, includingcovalent bonding either directly or via a linker group, and non-covalentassociations. Suitable covalent-bond carriers include proteins such asalbumins (e.g., U.S. Pat. No. 4,507,234), peptides, and polysaccharidessuch as aminodextran (e.g., U.S. Pat. No. 4,699,784), each of which havemultiple sites for the attachment of moieties. A carrier may also bearan agent by non-covalent associations, such as non-covalent bonding orby encapsulation, such as within a liposome vesicle (e.g., U.S. Pat.Nos. 4,429,008 and 4,873,088). Encapsulation carriers are especiallyuseful for imaging moiety conjugation to anti-T_(BT) antibody moietiesfor use in the invention, as a sufficient amount of the imaging moiety(dye, magnetic resonance contrast reagent, etc.) for detection may bemore easily associated with the antibody moiety. In addition,encapsulation carriers are also useful in chemotoxic therapeuticembodiments, as they can allow the therapeutic compositions to graduallyrelease a chemotoxic moiety over time while concentrating it in thevicinity of the tumor cells.

[0253] Carriers and linkers specific for radionuclide agents (both foruse as cytotoxic moieties or positron-emission imaging moieties) includeradiohalogenated small molecules and chelating compounds. For example,U.S. Pat. No. 4,735,792 discloses representative radiohalogenated smallmolecules and their synthesis. A radionuclide chelate may be formed fromchelating compounds that include those containing nitrogen and sulfuratoms as the donor atoms for binding the metal, or metal oxide,radionuclide. For example, U.S. Pat. No. 4,673,562, to Davison et al.discloses representative chelating compounds and their synthesis. Suchchelation carriers are also useful for magnetic spin contrast ions foruse in magnetic resonance imaging tumor visualization methods, and forthe chelation of heavy metal ions for use in radiographic visualizationmethods.

[0254] Preferred radionuclides for use as cytotoxic moieties areradionuclides which are suitable for pharmacological administration.Such radionuclides include ¹²³I, ¹²⁵I, ¹³¹I, ⁹⁰Y, ²¹¹At, ⁶⁷Cu, ¹⁸⁶Re,¹⁸⁸Re, ²¹²Pb, and ²¹²Bi. Iodine and astatine isotopes are more preferredradionuclides for use in the therapeutic compositions of the presentinvention, as a large body of literature has been accumulated regardingtheir use. ¹³¹I is particularly preferred, as are other β-radiationemitting nuclides, which have an effective range of several millimeters.¹²³I, ¹²⁵I, ¹³¹I, or ²¹¹At may be conjugated to antibody moieties foruse in the compositions and methods utilizing any of several knownconjugation reagents, including Iodogen, N-succinimidyl3-[²¹¹At]astatobenzoate, N-succinimidyl 3-[¹³³I]iodobenzoate (SIB), and,N-succinimidyl 5-[¹³¹I]iodob-3-pyridinecarboxylate (SIPC). Any iodineisotope may be utilized in the recited iodo-reagents. For example, asuitable antibody for use in the present invention may be easily made bycoupling an Fab fragment of the BD Transduction Labs R20720 anti-PTPζMAb with ¹³¹I Iodogen according to the manufacturer's instructions.Other radionuclides may be conjugated to anti-T_(BT)antibody moieties bysuitable chelation agents known to those of skill in the nuclearmedicine arts.

[0255] Preferred chemotoxic agents include small-molecule drugs such ascarboplatin, cisplatin, vincristine, taxanes such as paclitaxel anddoceltaxel, hydroxyurea, gemcitabine, vinorelbine, irinotecan,tirapazamine, matrilysin, methotrexate, pyrimidine and purine analogs,and other suitable small toxins known in the art. Preferred chemotoxindifferentiation inducers include phorbol esters and butyric acid.Chemotoxic moieties may be directly conjugated to theanti-T_(BT)antibody moiety via a chemical linker, or may encapsulated ina carrier, which is in turn coupled to the anti-T_(BT) antibody moiety.

[0256] Preferred toxin proteins for use as cytotoxic moieties includericins A and B, abrin, diphtheria toxin, bryodin 1 and 2, momordin,trichokirin, cholera toxin, gelonin, Pseudomonas exotoxin, Shigellatoxin, pokeweed antiviral protein, and other toxin proteins known in themedicinal biochemistry arts. As these toxin agents may elicitundesirable immune responses in the patient, especially if injectedintravascularly, it is preferred that they be encapsulated in a carrierfor coupling to the anti-T_(BT) antibody moiety.

[0257] Preferred radiographic moieties for use as imaging moieties inthe present invention include compounds and chelates with relativelylarge atoms, such as gold, iridium, technetium, barium, thallium,iodine, and their isotopes. It is preferred that less toxic radiographicimaging moieties, such as iodine or iodine isotopes, be utilized in thecompositions and methods of the invention. Examples of such compositionswhich may be utilized for x-ray radiography are described in U.S. Pat.No. 5,709,846, incorporated fully herein by reference. Such moieties maybe conjugated to the anti-T_(BT) antibody moiety through an acceptablechemical linker or chelation carrier. In addition, radionuclides whichemit radiation capable of penetrating the scull may be useful forscintillation imaging techniques. Suitable radionuclides for conjugationinclude ⁹⁹Tc, ¹¹¹In, and ⁶⁷Ga. Positron emitting moieties for use in thepresent invention include ¹⁸F, which can be easily conjugated by afluorination reaction with the anti-T_(BT) antibody moiety according tothe method described in U.S. Pat. No. 6,187,284.

[0258] Preferred magnetic resonance contrast moieties include chelatesof chromium(III), manganese(II), iron(II), nickel(II), copper(II),praseodymium(III), neodymium(III), samarium(III) and ytterbium(III) ion.Because of their very strong magnetic moment, the gadolinium(III),terbium(III), dysprosium(III), holmium(III), erbium(III), and iron(III)ions are especially preferred. Examples of such chelates, suitable formagnetic resonance spin imaging, are described in U.S. Pat. No.5,733,522, incorporated fully herein by reference. Nuclear spin contrastchelates may be conjugated to the anti-T_(BT) antibody moieties througha suitable chemical linker.

[0259] Optically visible moieties for use as imaging moieties includefluorescent dyes, or visible-spectrum dyes, visible particles, and othervisible labeling moieties. Fluorescent dyes such as fluorescein,coumarin, rhodamine, bodipy Texas red, and cyanine dyes, are useful whensufficient excitation energy can be provided to the site to be inspectedvisually. Endoscopic visualization procedures may be more compatiblewith the use of such labels. For many procedures where imaging agentsare useful, such as during an operation to resect a brain tumor, visiblespectrum dyes are preferred. Acceptable dyes include FDA-approved fooddyes and colors, which are non-toxic, although pharmaceuticallyacceptable dyes which have been approved for internal administration arepreferred. In preferred embodiments, such dyes are encapsulated incarrier moieties, which are in turn conjugated to the anti-T_(BT)antibody. Alternatively, visible particles, such as colloidal goldparticles or latex particles, may be coupled to the anti-T_(BT) antibodymoiety via a suitable chemical linker.

[0260] Delivery of Therapeutic and Imaging Agents to the Patient:

[0261] The Blood Brain Barrier (BBB) and Administration Strategies:

[0262] At one time, the BBB was not considered to present a problem inthe diagnosis and treatment of brain tumors, because early scans ofhuman brain tumors suggested that the BTB (blood tumor barrier) was“leaky.” However, as the size of the molecule increases, the rate ofmovement across the barrier decreases. The BBB has been demonstrated tobe heterogeneous in experimental human tumor xenograft animal models andin human patients. This lack of uniformity is because of the reducedintegrity of tight junctions in the capillary endothelial cells of thetumor neovasculature, intratumoral variation in permeability, andaltered intratumoral blood flow (Fuchs et al, 1990, Cancer research 50,1954-59, Groothuis et al., 1984, Prog. Exp. Tumor Res.) Thus, althoughthe BBB may not pose a delivery problem for some tumors in somepatients, this cannot be said for all brain tumors. In addition, apreferred mode of administration of the therapeutics of the invention isafter removal of the main tumor mass (resection of the tumor), whichdestroys much of the “leaky” neovasculature. Moreover, as braincarcinomas are usually pervasive throughout the organ, therapies whichare directed towards eradicating all tumor-producing cells cannot relyexclusively on the localized tumor neovasculature.

[0263] A first strategy for drug delivery through the BBB entailsdisruption of the BBB, either by osmotic means such as mannitol orleukotrienes, or biochemically by the use of vasoactive substances suchas bradykinin. The potential for using BBB opening to target specificagents to brain tumors is also an option. In preferred embodiments, aBBB disrupting agent is co-administered with the therapeutic or imagingcompositions of the invention when the compositions are administered byintravascular injection. Other strategies to go through the BBB mayentail the use of endogenous transport systems, includingcarrier-mediated transporters such as glucose and amino acid carriers,receptor-mediated transcytosis for insulin or transferrin, and activeefflux transporters such as p-glycoprotein. Active transport moietiesmay also be conjugated to the therapeutic or imaging compounds for usein the invention to facilitate transport across the epithelial wall ofthe blood vessel. However, the best current strategy for drug deliverybehind the BBB is by intrathecal delivery of therapeutics or imagingagents directly to the cranium, as through an Ommaya reservoir.

[0264] Delivery/Administration of Therapeutic Antibodies:

[0265] For administration, the antibody-therapeutic or antibody-imagingagent will generally be mixed, prior to administration, with anon-toxic, pharmaceutically acceptable carrier substance. Usually, thiswill be an aqueous solution, such as normal saline or phosphate-bufferedsaline (PBS), Ringer's solution, lactate-Ringer's solution, or anyisotonic physiologically acceptable solution for administration by thechosen means. Preferably, the solution is sterile and pyrogen-free, andis manufactured and packaged under current Good Manufacturing Processes(GMPs), as approved by the FDA. The clinician of ordinary skill isfamiliar with appropriate ranges for pH, tonicity, and additives orpreservatives when formulating pharmaceutical compositions foradministration by intravascular injection, intrathecal injection,injection into the cerebro-spinal fluid, direct injection into thetumor, or by other routes. In addition to additives for adjusting pH ortonicity, the antibody-therapeutics and antibody-imaging agents may bestabilized against aggregation and polymerization with amino acids andnon-ionic detergents, polysorbate, and polyethylene glycol. Optionally,additional stabilizers may include various physiologically-acceptablecarbohydrates and salts. Also, polyvinylpyrrolidone may be added inaddition to the amino acid. Suitable therapeutic immunoglobulinsolutions which are stabilized for storage and administration to humansare described in U.S. Pat. No. 5,945,098, incorporated fully herein byreference. Other agents, such as human serum albumin (HSA), may be addedto the therapeutic or imaging composition to stabilize the antibodyconjugates.

[0266] The compositions of the invention may be administered using anymedically appropriate procedure, e.g., intravascular (intravenous,intraarterial, intracapillary) administration, injection into thecerebrospinal fluid, intracavity or direct injection in the tumor.Intrathecal administration maybe carried out through the use of anOmmaya reservoir, in accordance with known techniques. (F. Balis et al.,Am J. Pediatr. Hematol. Oncol. 11, 74, 76 (1989). For the imagingcompositions of the invention, administration via intravascularinjection is preferred for pre-operative visualization of the tumor.Post-operative visualization or visualization concurrent with anoperation may be through intrathecal or intracavity administration, asthrough an Ommaya reservoir, or also by intravascular administration.

[0267] Intravascular injection may be by intravenous or intraarterialinjection: carotid artery injection is thought to assist inadministration to the brain, and is thus preferred. Antibody-agentsinjected into the blood stream have been shown to cross the blood-brainbarrier and to infiltrate the cranial cavity to some extent, usually inthe range of 10⁻⁴ to 10⁻³% [?UNITS?] injected dose per gram. This rateof uptake may be sufficient for imaging reagents, and also may be usefulfor tumor cell specific cytotoxic agents (e.g, those specificallydirected to the inhibition of the function of tumor-cell overexpressedproteins). However, in order to achieve therapeutic concentrations ofthe antibody-therapeutic agents without unacceptable toxicity to thepatient, it is preferred that the therapeutics compositions beadministered by intrathecal injection, direct injection, or injectioninto the cerebro-spinal fluid.

[0268] Thus, a preferred method for administration of the therapeuticcompositions of the invention is by depositing it into the inner cavityof a cystic tumor by any suitable technique, such as by direct injection(aided by stereotaxic positioning of an injection syringe, if necessary)or by placing the tip of an Ommaya reservoir into a cavity, or cyst, foradministration. Where the tumor is a solid tumor, the antibody may beadministered by first creating a resection cavity in the location of thetumor. This procedure differs from an ordinary craniotomy and tumorresection only in a few minor respects. As tumor resection is a commontreatment procedure, and is often indicated to relieve pressure,administration of the therapeutic compositions of the inventionfollowing tumor resection is a preferred embodiment of the treatmentmethods of the invention. Following gross total resection in a standardneurosurgical fashion, the cavity is preferable rinsed with saline untilall bleeding is stopped by cauterization. Next the pia-arachnoidmembrane, surrounding the tumor cavity at the surface, is cauterized toenhance the formation of fibroblastic reaction and scarring in thepia-arachnoid area. The result is the formation of an enclosed,fluid-filled cavity within the brain tissue at the location from wherethe tumor was removed. After the cyst has been formed, either the tip ofan Ommaya reservoir or a micro catheter, which is connected to a pumpdevice and allows the continues infusion of an antibody solution intothe cavity, can be placed into the cavity. See, e.g., U.S. Pat. No.5,558,852, incorporated fully herein by reference.

[0269] Alternatively, a convection-enhanced delivery catheter may beimplanted directly into the tumor mass, into a natural or surgicallycreated cyst, or into the normal brain mass. Such convection-enhancedpharmaceutical composition delivery devices greatly improve thediffusion of the composition throughout the brain mass. The implantedcatheters of these delivery devices utilize high-flow microinfusion(with flow rates in the range of about 0.5 to 15.0 μl/minute), ratherthan diffusive flow, to deliver the therapeutic or imaging compositionto the brain and/or tumor mass. Such devices are described in U.S. Pat.No. 5,720,720, incorporated fully herein by reference.

[0270] The effective amount of the therapeutic antibody-conjugatecomposition or of the imaging antibody-conjugate compositions to begiven to a particular patient will depend on a variety of factors,several of which will be different from patient to patient. A competentclinician will be able to determine an effective amount of a therapeuticantibody-conjugate composition to administer to a patient to retard thegrowth and promote the death of tumor cells, or an effective amount ofan imaging composition to administer to a patient to facilitate thevisualization of a tumor. Dosage of the antibody-conjugate will dependon the treatment of the tumor, route of administration, the nature ofthe therapeutics, sensitivity of the tumor to the therapeutics, etc.Utilizing LD₅₀ animal data, and other information available for theconjugated cytotoxic or imaging moiety, a clinician can determine themaximum safe dose for an individual, depending on the route ofadministration. For instance, an intravenously administered dose may bemore than an intrathecally administered dose, given the greater body offluid into which the therapeutic composition is being administered.Similarly, compositions which are rapidly cleared from the body may beadministered at higher doses, or in repeated doses, in order to maintaina therapeutic concentration. Imaging moieties are typically less toxicthan cytotoxic moieties and may be administered in higher doses in someembodiments. Utilizing ordinary skill, the competent clinician will beable to optimize the dosage of a particular therapeutic or imagingcomposition in the course of routine clinical trials.

[0271] Typically the dosage will be 0.001 to 100 milligrams of conjugateper kilogram subject body weight. Doses in the range of 0.01 to 1 mg perkilogram of patient body weight may be utilized for a radionuclidetherapeutic composition which is administered intrathecally. Relativelylarge doses, in the range of 0.1 to 10 mg per kilogram of patient bodyweight, may used for imaging conjugates with a relatively non-toxicimaging moiety. The amount utilized will depend on the sensitivity ofthe imaging method, and the relative toxicity of the imaging moiety. Ina therapeutic example, where the therapeutic composition comprises a¹³¹I cytotoxic moiety, the dosage to the patient will typically start ata lower range of 10 mCi, and go up to 100, 300 or even 500 mCi. Statedotherwise, where the therapeutic agent is ¹³¹I, the dosage to thepatient will typically be from 5,000 Rads to 100,000 Rads (preferably atleast 13,000 Rads, or even at least 50,000 Rads). Doses for otherradionuclides are typically selected so that the tumoricidal dose willbe equivalent to the foregoing range for ¹³¹I. Similarly, chemotoxic ortoxin protein doses may be scaled accordingly.

[0272] The antibody conjugate can be administered to the subject in aseries of more than one administration. For therapeutic compositions,regular periodic administration (e.g., every 2-3 days) will sometimes berequired, or may be desirable to reduce toxicity. For therapeuticcompositions which will be utilized in repeated-dose regimens, antibodymoieties which do not provoke HAMA or other immune responses arepreferred. The imaging antibody conjugate compositions may beadministered at an appropriate time before the visualization technique.For example, administration within an hour before direct visualinspection may be appropriate, or administration within twelve hoursbefore an MRI scan may be appropriate. Care should be taken, however, tonot allow too much time to pass between administration andvisualization, as the imaging compound may eventually be cleared fromthe patient's system.

[0273] In addition to the use of imaging antibody conjugates for simplevisualization, these compositions may be utilized as a “dry run” formore toxic cytotoxic antibody conjugates. If the same antibody moiety isutilized for the imaging conjugate as for the therapeutic conjugate, thephysician may first use a visualization technique to determine preciselywhere in the brain the cytotoxic conjugate will concentrate. If asufficient degree of tissue selectivity is not achieved (e.g, if thetumor cells are too disperse in the normal tissue, or if the particularbrain tumor protein target chosen is not sufficiently overexpressed inthe particular patient's tumor cells), then the physician may chooseanother brain tumor protein target. The provision of numerous braintumor protein targets by the present invention, along with both imagingand therapeutic agents, allows a high degree of flexibility in designingan effective treatment regimen for the individual patient.

[0274] Combination Therapies of the Invention

[0275] As mentioned previously, brain tumors tend to be heterogeneous incharacter, and pervasive throughout the brain tissue. This combinationoften makes them difficult to treat, as individual portions of the tumorcells in any particular patient may have differing biologicalcharacteristic. Thus, in some cases, it may be preferred to use variouscombinations of therapeutic or imaging agents, as described above in theSummary of Invention, in order to more fully target all of the cellsexhibiting tumorigenic characteristics. Such combination treatments maybe by administering blended antibody therapeutic or imagingcompositions, individually prepared as described above, andadministering the blended therapeutic to the patient as described. Theskilled administering physician will be able to take such factors ascombined toxicity, and individual antibody agent efficacy, into accountwhen administering such combined agents. Additionally, those of skill inthe art will be able to screen the antibodies to avoid potentialcross-reaction with each other, in order to assure full efficacy of eachantibody therapeutic or imaging agent.

[0276] Alternatively, several individual brain tumor protein targetcompositions may be administered simultaneously or in succession for acombined therapy. This may be desirable to avoid accumulated toxicityfrom several antibody conjugate reagents, or to more closely monitorpotential adverse reactions to the individual antibody reagents. Thus,cycles such as where a first antibody therapeutic agent is administeredon day one, followed by a second on day two, then a period with outadministration, followed by re-administration of the antibodytherapeutics on different successive days, is comprehended within thepresent invention.

EXAMPLES Example 1 Identification of Two New Splicing Variant Isoformsof PTPζ: PTPζ SM1 and SM2

[0277] The mRNA nucleotide sequence for PTPζ SM1 was identified in ahuman fetal brain phage cDNA library by sequencing.

[0278] The mRNA nucleotide sequence for PTPζ SM2 was identified by PCRamplification of adult human brain cDNA, and sequencing of the resultingnucleic acids.

[0279] For the RT-PCR analyses performed below, total RNA was isolatedfrom either cells (glioblastoma cultured lines) or tissue using Trizole(Gibco Life Technologies, Inc.), following the manufacture's protocol.cDNA was generated from total RNA using the 1^(st) Strand synthesis kitfrom Gibco Life Technologies, Inc., and an oligo dT₃₀ anchored primer.For each RT-PCR reaction, 1 μl of cDNA was utilized. The PCR reactionwas carried out using an Advantage 2 kit (Clontech) under standardconditions. The products of the PCR reactions were confirmed viasequencing.

[0280] Both clones were verified by RT-PCR analysis of glioblastoma celllines and primary tumors. For PTPζ SM1, primers CAGCAGTTGGATGGAAGAGGAC[SEQ ID NO. 28] and CACTGAGATTCTGGCACTATTC [SEQ ID NO. 29] were used,producing an identifiable 1116 bp product. RT-PCR analysis wasperformed, confirming expression of the SM1 splice variant in 11 of 17different glioblastoma cell lines tested, fetal brain, and adult brain,using the unique 3 end and portion of the 3′ untranslated region as thehybridization target for the probe. In addition, RT-PCR analysis wasperformed on 28 primary brain tumor samples, confirming expression ofthe PTPζ SM1 variant in 16 of the 28 tumors.

[0281] For PTPζ SM2, primers AACAATTCCAGGGTCTCACTC [SEQ ID NO. 30] andTTGACTGGCTCAGGAGTATAG [SEQ ID NO. 31] were used, which produce a 130 bpproduct when the extra exon 23a is present, and a no product when theexon 23a is absent. RT-PCR analysis was performed, confirming expressionin 6 of 17 different glioblastoma cell lines tested. In addition, RT-PCRanalysis was performed on 28 primary brain tumor samples, confirmingexpression of the PTPζ SM1 variant in 19 of the 28 tumors.

[0282] For comparison, RT-PCR analysis was also done for the expressionof PTPζ-α (primers CTGATAATGAGGGCTCCCAAC [SEQ ID NO. 32] andCTCTGCACTTCCTGGTAAAACTCT [SEQ ID NO. 33]) and PTPζ-β (primersCAGCAGTTGGATGGAAGAGGAC [SEQ ID NO. 34] and CTCTGCACTTCCTGGTAAAACTCT [SEQID NO. 35]) in the 28 brain tumor tissue samples. PTPζ-α was shown to beexpressed in 16 of the 28 samples, and the short form PTPζ-β was shownto be expressed in 19 of the 28 samples.

[0283] The nucleotide sequence alignment of the two new splice variantswith the reference sequence for PTPζ-α is shown in the following table:TABLE 2 PTP 5′ PTP 3′ PAC 1 5′ PAC 1 3′ Corresponding Exon Key: 1 4887274 87321 5′ UTR PAC 1: RPS-1062J16 BAC: RP11-384A20 70 205 8734387487  1 PAC 2: RPS-1049N15 205 272 142076 142143  2 BAC 5′ BAC 3′ 291451 24001 24161 *  3 *88 nt deletion seen in 5′ PCR clone from PTP363-451 450 603 28570 28723  4 602 701 32814 32888  5 698 772 3281432888  6 766 924 39695 39853  7 922 1075 39995 40148  8 1074 1261 5241152598 *  9 *not spliced at 1261 in phage library clones 1260 1387 5391054037 10 1387 1435 60644 60692 11 1432 2346 66362 67276  5′ 12(end ofBAC) PAC 2 5′ PAC 2 3′ 2147 4409 1 2263 mid 12 4437 4987 2294 2844  3′12 4925 5133 8027 8224 13 5131 5224 17505 17598 14 5223 5310 20427 2051415 5309 5332 23048 23071 16 5329 5428 23234 23333 17 5429 5512 2555525638 18 5512 5646 27710 27844 19 5572 5602 42925 42955 * Duplicate ofmid 19 *duplicated regions of exons 19 5646 5768 28408 28530 most of 20(−12 bp 3′) and 26 vary by one aa/two nt 5791 5945 29770 29934 21 (−10bp 5′) 5943 6082 31560 31699 22 6080 6228 33375 33523 ˜ 23 ˜116 ntinsert seen b/w exons 23 & 24 in 3′ PCR clone: maps to PAC b/w 23 & 246225 6322 40379 40476 ˜ 24 PTP location PAC 2 5′ PAC 2 3′ 6322 639740820 40895 25 6228 36744 36629 6396 6526 42864 42994 26 6457 6487 2777027800 * Duplicate of mid 26 6525 6673 43895 44043 27 6671 6816 4775347898 28 6816 6952 48708 48844 29

Example 2 Cell Migration Assay For Determining Antibody Activity onProtein Targets

[0284] Tumor cells are known to migrate more rapidly towardschemoattractants. The cell migration assay measures the ability of acell to migrate. The ability to migrate is taken as a measure oftumorigenicity. Chemoattractants generally used are fetal bovine serum,pleiotrophin, bFGF, and VEGF. Thus, this assay can be used to determinemigration capability of a cell in which the gene has been knocked downor the gene of interest is being overexpressed.

[0285] The ChemoTx® disposable chemotaxis system (Neuroprobe, Inc.,Gaithersburg, Md.) is used according to the manufacturer's instructions,with a few modifications. Briefly, glioblastoma cultured cells from cellline G55T2 are prepared by splitting the cells the day before the assayis performed. A ChemoTx® chamber with the following specifications isused: Pore size 8 μm, exposed filter area 8 mm, exposed filter areadiameter 3.2 mm². The plate configuration is: 30 μl per well, 96 wellplate. The membrane type is: Track-etched polycarbonate.

[0286] In preparation for the assays, the filter membrane is coated in100 ml PBS containing 0.1% acetic acid and 3.5 ml Vitrogen 100 (fromCohesion) at 37° C. overnight. About 30 minutes before starting theassay the coated membrane is washed and rinsed with PBS containing 0.1%BSA. Cells are harvested by using the standard technique (trypsin-EDTA).The cells are washed once with DMEM 10% FBS, and then spun at 1000 RPM,for 5 minutes at room temperature. The pellet is resuspended in DMEMwithout serum, containing 0.1% BSA (serum free medium). The cells arespun and resuspended again in serum free medium, and then spun andresuspended in the amount of serum free medium needed to provide aconcentration of 1 mio. cells/ml, or 25,000 cells per 25 ul. Just priorto the assay, a suitable amount of the antibody to be tested foranti-target function activity is added to the cell suspension.

[0287] For the assay, a standard chemoattractant is used to measure themobility of the cells. The chemoattractants are diluted in serum freemedium. A suitable unspecific chemoattractant is DMEM with 5% FBS. Thechemoattractant solutions and control solutions without chemoattractantare pipetted (29 μl) into the lower plate wells. After placing andsecuring the filter plate over the lower wells, ensuring contact withthe solution in the bottom wells, serial dilutions of the cellsuspension are pipetted onto each site on the filter top. The plates arethem covered and incubated at 37° C., 5% CO₂, for 3-4 hours.

[0288] After incubation, the upper filter side is rinsed with PBS andexposed upper filter areas are cleaned with wet cotton swabs. The filteris stained using Diff-Quik™ (VWR) dye kit, according to themanufacturer's instructions. The migrated cells are counted on the lowerfilter side using a microscope (Magnification 200×), by counting of 5high power field sections per well.

Example 2 HUVEC(Human Umbilical Vein Endothelial Cells) EndothelialSprouting Assay For Determining Antibody Activity on Protein Targets

[0289] Cell-sprouting morphology can be utilized as an easily visualizedassay to determine the inhibitory effect of a candidate antibody on theprotein target function for protein targets which stimulate endothelialcell sprouting, such as ARP2. Such assays have been describedextensively in the literature (Nehls, V., et al., Histochem. Cell Biol.104: 459-466 (1995); Koblizek, T. I., et al., Curr. Biol. 8: 529-532(1988); and Kwak, H. J., et al., FEBS Lett. 448: 249-253). Briefly, aendothelial cells from a suitable source, such as HUVECs or PPAECs(porcine pulmonary artery endothelial cells) are grown to confluence onmicrocarrier (MC) beads (diameter 175 μm, available from Sigma) andplaced into a 2.5 mg/ml fibrinogen gel containing the protein target atan appropriate effective concentration (200 ng/ml is an suitablestarting concentration, which the skilled practitioner may optimize) andthe antibody in an appropriate range of concentrations (this will dependon antibody titer and affinity for the target), and 200 units/mlTrasylol (available from Bayer). Fibrin gels are incubated in M-199 witha daily supplement of the same amount of recombinant protein andantibody, 2.0% heat-inactivated fetal bovine serum, and 200 units/mlTrasylol. After three days, the extent of sprouting is determined usinga phase-contrast microscope (such as those available from Zeiss). Adecrease in cell sprouting as compared to controls without antibodyindicates a reduction in protein target activity by the antibody.

[0290] The foregoing is intended to be illustrative of the embodimentsof the present invention, and are not intended to limit the invention inany way. Although the invention has been described with respect tospecific modifications, the details thereof are not to be construed aslimitations, for it will be apparent that various equivalents, changesand modifications may be resorted to without departing from the spiritand scope thereof and it is understood that such equivalent embodimentsare to be included herein. All publications and patent applications areherein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

1 35 1 3091 DNA Homo sapiens gene (1)..(3091) PTP-zeta SM1 exon 9variant 1 cacacatacg cacgcacgat ctcacttcga tctatacact ggaggattaaaacaaacaaa 60 caaaaaaaac atttccttcg ctccccctcc ctctccactc tgagaagcagaggagccgca 120 cggcgagggg ccgcagaccg tctggaa atg cga atc cta aag cgt ttcctc gct 174 Met Arg Ile Leu Lys Arg Phe Leu Ala 1 5 tgc att cag ctc ctctgt gtt tgc cgc ctg gat tgg gct aat gga tac 222 Cys Ile Gln Leu Leu CysVal Cys Arg Leu Asp Trp Ala Asn Gly Tyr 10 15 20 25 tac aga caa cag agaaaa ctt gtt gaa gag att ggc tgg tcc tat aca 270 Tyr Arg Gln Gln Arg LysLeu Val Glu Glu Ile Gly Trp Ser Tyr Thr 30 35 40 gga gca ctg aat caa aaaaat tgg gga aag aaa tat cca aca tgt aat 318 Gly Ala Leu Asn Gln Lys AsnTrp Gly Lys Lys Tyr Pro Thr Cys Asn 45 50 55 agc cca aaa caa tct cct atcaat att gat gaa gat ctt aca caa gta 366 Ser Pro Lys Gln Ser Pro Ile AsnIle Asp Glu Asp Leu Thr Gln Val 60 65 70 aat gtg aat ctt aag aaa ctt aaattt cag ggt tgg gat aaa aca tca 414 Asn Val Asn Leu Lys Lys Leu Lys PheGln Gly Trp Asp Lys Thr Ser 75 80 85 ttg gaa aac aca ttc att cat aac actggg aaa aca gtg gaa att aat 462 Leu Glu Asn Thr Phe Ile His Asn Thr GlyLys Thr Val Glu Ile Asn 90 95 100 105 ctc act aat gac tac cgt gtc agcgga gga gtt tca gaa atg gtg ttt 510 Leu Thr Asn Asp Tyr Arg Val Ser GlyGly Val Ser Glu Met Val Phe 110 115 120 aaa gca agc aag ata act ttt cactgg gga aaa tgc aat atg tca tct 558 Lys Ala Ser Lys Ile Thr Phe His TrpGly Lys Cys Asn Met Ser Ser 125 130 135 gat gga tca gag cat agt tta gaagga caa aaa ttt cca ctt gag atg 606 Asp Gly Ser Glu His Ser Leu Glu GlyGln Lys Phe Pro Leu Glu Met 140 145 150 caa atc tac tgc ttt gat gcg gaccga ttt tca agt ttt gag gaa gca 654 Gln Ile Tyr Cys Phe Asp Ala Asp ArgPhe Ser Ser Phe Glu Glu Ala 155 160 165 gtc aaa gga aaa ggg aag tta agagct tta tcc att ttg ttt gag gtt 702 Val Lys Gly Lys Gly Lys Leu Arg AlaLeu Ser Ile Leu Phe Glu Val 170 175 180 185 ggg aca gaa gaa aat ttg gatttc aaa gcg att att gat gga gtc gaa 750 Gly Thr Glu Glu Asn Leu Asp PheLys Ala Ile Ile Asp Gly Val Glu 190 195 200 agt gtt agt cgt ttt ggg aagcag gct gct tta gat cca ttc ata ctg 798 Ser Val Ser Arg Phe Gly Lys GlnAla Ala Leu Asp Pro Phe Ile Leu 205 210 215 ttg aac ctt ctg cca aac tcaact gac aag tat tac att tac aat ggc 846 Leu Asn Leu Leu Pro Asn Ser ThrAsp Lys Tyr Tyr Ile Tyr Asn Gly 220 225 230 tca ttg aca tct cct ccc tgcaca gac aca gtt gac tgg att gtt ttt 894 Ser Leu Thr Ser Pro Pro Cys ThrAsp Thr Val Asp Trp Ile Val Phe 235 240 245 aaa gat aca gtt agc atc tctgaa agc cag ttg gct gtt ttt tgt gaa 942 Lys Asp Thr Val Ser Ile Ser GluSer Gln Leu Ala Val Phe Cys Glu 250 255 260 265 gtt ctt aca atg caa caatct ggt tat gtc atg ctg atg gac tac tta 990 Val Leu Thr Met Gln Gln SerGly Tyr Val Met Leu Met Asp Tyr Leu 270 275 280 caa aac aat ttt cga gagcaa cag tac aag ttc tct aga cag gtg ttt 1038 Gln Asn Asn Phe Arg Glu GlnGln Tyr Lys Phe Ser Arg Gln Val Phe 285 290 295 tcc tca tac act gga aaggaa gag att cat gaa gca gtt tgt agt tca 1086 Ser Ser Tyr Thr Gly Lys GluGlu Ile His Glu Ala Val Cys Ser Ser 300 305 310 gaa cca gaa aat gtt caggct gac cca gag aat tat acc agc ctt ctt 1134 Glu Pro Glu Asn Val Gln AlaAsp Pro Glu Asn Tyr Thr Ser Leu Leu 315 320 325 gtt aca tgg gaa aga cctcga gtc gtt tat gat acc atg att gag aag 1182 Val Thr Trp Glu Arg Pro ArgVal Val Tyr Asp Thr Met Ile Glu Lys 330 335 340 345 ttt gca gtt ttg taccag cag ttg gat gga gag gac caa acc aag cat 1230 Phe Ala Val Leu Tyr GlnGln Leu Asp Gly Glu Asp Gln Thr Lys His 350 355 360 gaa ttt ttg aca gatggc tat caa gac ttg gta act ata tga 1272 Glu Phe Leu Thr Asp Gly Tyr GlnAsp Leu Val Thr Ile 365 370 tcagttgttt tacatagggt aacattataa tttaatttccaaggtaagaa cttacaaatg 1332 gttgtatatt attttcctcc attactttta gactttatgtgaaggtgggg taggctgagt 1392 atttttaaat ttaaaaaaaa attttaaatt agaagctatactaaattatg tttaaagtta 1452 catttaatta aatggatatc ataactttgc caacaataacactatagagt agatacatat 1512 gacttatgaa ctggagatca tttagtgtgg cctttcttaagatttcagtt gtagaatagt 1572 gccagaatct cagtgccctg atacatttta tattgtgtcttccattacgc tatatcagca 1632 caggaaaagt agagtagggg acatacaagt cctctttgttgcaccaaaaa attttcagat 1692 aacagctggg aagtcatgat tgggtcagaa ctttggggatgtaagaaaac atttcttaca 1752 aaaagatcca cccctgcctc cctccaccag cgcatgcgaataaagtacag attccctttg 1812 tggcctgagc atgtcagtat taaactttgc tctggtagggaagtgttggc catagattag 1872 ggtgtagttg acaaaccttc atctggatgt aggtccagaaagtccccact gcaggttaaa 1932 ggacactgga ctctgcactc aggcacctag agtcctgcaagtcctgggaa cctgcattta 1992 aataaaaatg cactattaat tatgtttcat atcatgtggacaaaatggat aaaattttag 2052 taacctttta attcagttgc ctggaatatg gagacacaatgacctgggaa aatcgtgaaa 2112 taaatagtaa taaaaatgtt tatttcataa ttacgtgaagaagataattc tattactgtt 2172 cttgcatata tattgtcaag aaaaagagat aacttagttgttcacttttt cacattgctc 2232 cttgtttgca aatgcccccc atttatttgt ctaaaatattaatttttagt ttgtagtact 2292 aatttatgaa tttgatgagt tctggctaaa aatgaaacttcctgaaacta aatctgattt 2352 ttaaaaagca aaaaaaaaaa aaaagcctag ctttccagttcttcataatt cacaaatacc 2412 acaagtttaa ctaagcaaca ttgcataaac ttttccttaggttaataaaa tagaagtatt 2472 ttccacggac cagggagaaa aagttttcta ggaaagatacctagtgtgtt ggtagtccta 2532 tgagaataac atttgtataa ttactaacat ctttcttttagggtgctatt ctcaataatt 2592 tgctacccaa tatgagttat gttcttcaga tagtagccatatgcactaat ggcttatatg 2652 gaaaatacag cgaccaactg attgtcgaca tgcctactgataatcctggt aagtgccacc 2712 agatacatct atatattaac tcaataaatg aggttagtttaattactgta tgcattgatg 2772 ctttctctct atattctttt ggccaaaagg caaagtgattttctcttaag tctggattgc 2832 cgggtaattt tttggggcat gggacccatt tctcattcagcaggtctggt gccagacaat 2892 aagtaaactt atccttaata ttggagttta ccatttgtaaaataagagtg actaaacata 2952 tttataacat tgtaataatc attaaatgaa aattgctatgtaaatgttga gactgttatt 3012 ttggataatt aagagttggt ttaatttgta tttatttcctcttttcagcc cccaaagcat 3072 tatgtagtaa gtgtataca 3091 2 374 PRT Homosapiens gene (1)..(374) PTP-zeta SM1 exon 9 variant 2 Met Arg Ile LeuLys Arg Phe Leu Ala Cys Ile Gln Leu Leu Cys Val 1 5 10 15 Cys Arg LeuAsp Trp Ala Asn Gly Tyr Tyr Arg Gln Gln Arg Lys Leu 20 25 30 Val Glu GluIle Gly Trp Ser Tyr Thr Gly Ala Leu Asn Gln Lys Asn 35 40 45 Trp Gly LysLys Tyr Pro Thr Cys Asn Ser Pro Lys Gln Ser Pro Ile 50 55 60 Asn Ile AspGlu Asp Leu Thr Gln Val Asn Val Asn Leu Lys Lys Leu 65 70 75 80 Lys PheGln Gly Trp Asp Lys Thr Ser Leu Glu Asn Thr Phe Ile His 85 90 95 Asn ThrGly Lys Thr Val Glu Ile Asn Leu Thr Asn Asp Tyr Arg Val 100 105 110 SerGly Gly Val Ser Glu Met Val Phe Lys Ala Ser Lys Ile Thr Phe 115 120 125His Trp Gly Lys Cys Asn Met Ser Ser Asp Gly Ser Glu His Ser Leu 130 135140 Glu Gly Gln Lys Phe Pro Leu Glu Met Gln Ile Tyr Cys Phe Asp Ala 145150 155 160 Asp Arg Phe Ser Ser Phe Glu Glu Ala Val Lys Gly Lys Gly LysLeu 165 170 175 Arg Ala Leu Ser Ile Leu Phe Glu Val Gly Thr Glu Glu AsnLeu Asp 180 185 190 Phe Lys Ala Ile Ile Asp Gly Val Glu Ser Val Ser ArgPhe Gly Lys 195 200 205 Gln Ala Ala Leu Asp Pro Phe Ile Leu Leu Asn LeuLeu Pro Asn Ser 210 215 220 Thr Asp Lys Tyr Tyr Ile Tyr Asn Gly Ser LeuThr Ser Pro Pro Cys 225 230 235 240 Thr Asp Thr Val Asp Trp Ile Val PheLys Asp Thr Val Ser Ile Ser 245 250 255 Glu Ser Gln Leu Ala Val Phe CysGlu Val Leu Thr Met Gln Gln Ser 260 265 270 Gly Tyr Val Met Leu Met AspTyr Leu Gln Asn Asn Phe Arg Glu Gln 275 280 285 Gln Tyr Lys Phe Ser ArgGln Val Phe Ser Ser Tyr Thr Gly Lys Glu 290 295 300 Glu Ile His Glu AlaVal Cys Ser Ser Glu Pro Glu Asn Val Gln Ala 305 310 315 320 Asp Pro GluAsn Tyr Thr Ser Leu Leu Val Thr Trp Glu Arg Pro Arg 325 330 335 Val ValTyr Asp Thr Met Ile Glu Lys Phe Ala Val Leu Tyr Gln Gln 340 345 350 LeuAsp Gly Glu Asp Gln Thr Lys His Glu Phe Leu Thr Asp Gly Tyr 355 360 365Gln Asp Leu Val Thr Ile 370 3 8058 DNA Homo sapiens gene (1)..(8058)PTP-zeta SM2 exon 23a variant 3 cacacatacg cacgcacgat ctcacttcgatctatacact ggaggattaa aacaaacaaa 60 caaaaaaaac atttccttcg ctccccctccctctccactc tgagaagcag aggagccgca 120 cggcgagggg ccgcagaccg tctggaaatgcgaatcctaa agcgtttcct cgcttgcatt 180 cagctcctct gtgtttgccg cctggattgggctaatggat actacagaca acagagaaaa 240 cttgttgaag agattggctg gtcctatacaggagcactga atcaaaaaaa ttggggaaag 300 aaatatccaa catgtaatag cccaaaacaatctcctatca atattgatga agatcttaca 360 caagtaaatg tgaatcttaa gaaacttaaatttcagggtt gggataaaac atcattggaa 420 aacacattca ttcataacac tgggaaaacagtggaaatta atctcactaa tgactaccgt 480 gtcagcggag gagtttcaga aatggtgtttaaagcaagca agataacttt tcactgggga 540 aaatgcaata tgtcatctga tggatcagagcatagtttag aaggacaaaa atttccactt 600 gagatgcaaa tctactgctt tgatgcggaccgattttcaa gttttgagga agcagtcaaa 660 ggaaaaggga agttaagagc tttatccattttgtttgagg ttgggacaga agaaaatttg 720 gatttcaaag cgattattga tggagtcgaaagtgttagtc gttttgggaa gcaggctgct 780 ttagatccat tcatactgtt gaaccttctgccaaactcaa ctgacaagta ttacatttac 840 aatggctcat tgacatctcc tccctgcacagacacagttg actggattgt ttttaaagat 900 acagttagca tctctgaaag ccagttggctgttttttgtg aagttcttac aatgcaacaa 960 tctggttatg tcatgctgat ggactacttacaaaacaatt ttcgagagca acagtacaag 1020 ttctctagac aggtgttttc ctcatacactggaaaggaag agattcatga agcagtttgt 1080 agttcagaac cagaaaatgt tcaggctgacccagagaatt ataccagcct tcttgttaca 1140 tgggaaagac ctcgagtcgt ttatgataccatgattgaga agtttgcagt tttgtaccag 1200 cagttggatg gagaggacca aaccaagcatgaatttttga cagatggcta tcaagacttg 1260 ggtgctattc tcaataattt gctacccaatatgagttatg ttcttcagat agtagccata 1320 tgcactaatg gcttatatgg aaaatacagcgaccaactga ttgtcgacat gcctactgat 1380 aatcctgaac ttgatctttt ccctgaattaattggaactg aagaaataat caaggaggag 1440 gaagagggaa aagacattga agaaggcgctattgtgaatc ctggtagaga cagtgctaca 1500 aaccaaatca ggaaaaagga accccagatttctaccacaa cacactacaa tcgcataggg 1560 acgaaataca atgaagccaa gactaaccgatccccaacaa gaggaagtga attctctgga 1620 aagggtgatg ttcccaatac atctttaaattccacttccc aaccagtcac taaattagcc 1680 acagaaaaag atatttcctt gacttctcagactgtgactg aactgccacc tcacactgtg 1740 gaaggtactt cagcctcttt aaatgatggctctaaaactg ttcttagatc tccacatatg 1800 aacttgtcgg ggactgcaga atccttaaatacagtttcta taacagaata tgaggaggag 1860 agtttattga ccagtttcaa gcttgatactggagctgaag attcttcagg ctccagtccc 1920 gcaacttctg ctatcccatt catctctgagaacatatccc aagggtatat attttcctcc 1980 gaaaacccag agacaataac atatgatgtccttataccag aatctgctag aaatgcttcc 2040 gaagattcaa cttcatcagg ttcagaagaatcactaaagg atccttctat ggagggaaat 2100 gtgtggtttc ctagctctac agacataacagcacagcccg atgttggatc aggcagagag 2160 agctttctcc agactaatta cactgagatacgtgttgatg aatctgagaa gacaaccaag 2220 tccttttctg caggcccagt gatgtcacagggtccctcag ttacagatct ggaaatgcca 2280 cattattcta cctttgccta cttcccaactgaggtaacac ctcatgcttt taccccatcc 2340 tccagacaac aggatttggt ctccacggtcaacgtggtat actcgcagac aacccaaccg 2400 gtatacaatg gtgagacacc tcttcaaccttcctacagta gtgaagtctt tcctctagtc 2460 acccctttgt tgcttgacaa tcagatcctcaacactaccc ctgctgcttc aagtagtgat 2520 tcggccttgc atgctacgcc tgtatttcccagtgtcgatg tgtcatttga atccatcctg 2580 tcttcctatg atggtgcacc tttgcttccattttcctctg cttccttcag tagtgaattg 2640 tttcgccatc tgcatacagt ttctcaaatccttccacaag ttacttcagc taccgagagt 2700 gataaggtgc ccttgcatgc ttctctgccagtggctgggg gtgatttgct attagagccc 2760 agccttgctc agtattctga tgtgctgtccactactcatg ctgcttcaga gacgctggaa 2820 tttggtagtg aatctggtgt tctttataaaacgcttatgt tttctcaagt tgaaccaccc 2880 agcagtgatg ccatgatgca tgcacgttcttcagggcctg aaccttctta tgccttgtct 2940 gataatgagg gctcccaaca catcttcactgtttcttaca gttctgcaat acctgtgcat 3000 gattctgtgg gtgtaactta tcagggttccttatttagcg gccctagcca tataccaata 3060 cctaagtctt cgttaataac cccaactgcatcattactgc agcctactca tgccctctct 3120 ggtgatgggg aatggtctgg agcctcttctgatagtgaat ttcttttacc tgacacagat 3180 gggctgacag cccttaacat ttcttcacctgtttctgtag ctgaatttac atatacaaca 3240 tctgtgtttg gtgatgataa taaggcgctttctaaaagtg aaataatata tggaaatgag 3300 actgaactgc aaattccttc tttcaatgagatggtttacc cttctgaaag cacagtcatg 3360 cccaacatgt atgataatgt aaataagttgaatgcgtctt tacaagaaac ctctgtttcc 3420 atttctagca ccaagggcat gtttccagggtcccttgctc ataccaccac taaggttttt 3480 gatcatgaga ttagtcaagt tccagaaaataacttttcag ttcaacctac acatactgtc 3540 tctcaagcat ctggtgacac ttcgcttaaacctgtgctta gtgcaaactc agagccagca 3600 tcctctgacc ctgcttctag tgaaatgttatctccttcaa ctcagctctt attttatgag 3660 acctcagctt cttttagtac tgaagtattgctacaacctt cctttcaggc ttctgatgtt 3720 gacaccttgc ttaaaactgt tcttccagctgtgcccagtg atccaatatt ggttgaaacc 3780 cccaaagttg ataaaattag ttctacaatgttgcatctca ttgtatcaaa ttctgcttca 3840 agtgaaaaca tgctgcactc tacatctgtaccagtttttg atgtgtcgcc tacttctcat 3900 atgcactctg cttcacttca aggtttgaccatttcctatg caagtgagaa atatgaacca 3960 gttttgttaa aaagtgaaag ttcccaccaagtggtacctt ctttgtacag taatgatgag 4020 ttgttccaaa cggccaattt ggagattaaccaggcccatc ccccaaaagg aaggcatgta 4080 tttgctacac ctgttttatc aattgatgaaccattaaata cactaataaa taagcttata 4140 cattccgatg aaattttaac ctccaccaaaagttctgtta ctggtaaggt atttgctggt 4200 attccaacag ttgcttctga tacatttgtatctactgatc attctgttcc tataggaaat 4260 gggcatgttg ccattacagc tgtttctccccacagagatg gttctgtaac ctcaacaaag 4320 ttgctgtttc cttctaaggc aacttctgagctgagtcata gtgccaaatc tgatgccggt 4380 ttagtgggtg gtggtgaaga tggtgacactgatgatgatg gtgatgatga tgatgacaga 4440 gatagtgatg gcttatccat tcataagtgtatgtcatgct catcctatag agaatcacag 4500 gaaaaggtaa tgaatgattc agacacccacgaaaacagtc ttatggatca gaataatcca 4560 atctcatact cactatctga gaattctgaagaagataata gagtcacaag tgtatcctca 4620 gacagtcaaa ctggtatgga cagaagtcctggtaaatcac catcagcaaa tgggctatcc 4680 caaaagcaca atgatggaaa agaggaaaatgacattcaga ctggtagtgc tctgcttcct 4740 ctcagccctg aatctaaagc atgggcagttctgacaagtg atgaagaaag tggatcaggg 4800 caaggtacct cagatagcct taatgagaatgagacttcca cagatttcag ttttgcagac 4860 actaatgaaa aagatgctga tgggatcctggcagcaggtg actcagaaat aactcctgga 4920 ttcccacagt ccccaacatc atctgttactagcgagaact cagaagtgtt ccacgtttca 4980 gaggcagagg ccagtaatag tagccatgagtctcgtattg gtctagctga ggggttggaa 5040 tccgagaaga aggcagttat accccttgtgatcgtgtcag ccctgacttt tatctgtcta 5100 gtggttcttg tgggtattct catctactggaggaaatgct tccagactgc acacttttac 5160 ttagaggaca gtacatcccc tagagttatatccacacctc caacacctat ctttccaatt 5220 tcagatgatg tcggagcaat tccaataaagcactttccaa agcatgttgc agatttacat 5280 gcaagtagtg ggtttactga agaatttgagacactgaaag agttttacca ggaagtgcag 5340 agctgtactg ttgacttagg tattacagcagacagctcca accacccaga caacaagcac 5400 aagaatcgat acataaatat cgttgcctatgatcatagca gggttaagct agcacagctt 5460 gctgaaaagg atggcaaact gactgattatatcaatgcca attatgttga tggctacaac 5520 agaccaaaag cttatattgc tgcccaaggcccactgaaat ccacagctga agatttctgg 5580 agaatgatat gggaacataa tgtggaagttattgtcatga taacaaacct cgtggagaaa 5640 ggaaggagaa aatgtgatca gtactggcctgccgatggga gtgaggagta cgggaacttt 5700 ctggtcactc agaagagtgt gcaagtgcttgcctattata ctgtgaggaa ttttactcta 5760 agaaacacaa aaataaaaaa gggctcccagaaaggaagac ccagtggacg tgtggtcaca 5820 cagtatcact acacgcagtg gcctgacatgggagtaccag agtactccct gccagtgctg 5880 acctttgtga gaaaggcagc ctatgccaagcgccatgcag tggggcctgt tgtcgtccac 5940 tgcagtgctg gagttggaag aacaggcacatatattgtgc tagacagtat gttgcagcag 6000 attcaacacg aaggaactgt caacatatttggcttcttaa aacacatccg ttcacaaaga 6060 aattatttgg tacaaactga ggagcaatatgtcttcattc atgatacact ggttgaggcc 6120 atacttagta aagaaactga ggtgctggacagtcatattc atgcctatgt taatgcactc 6180 ctcattcctg gaccagcagg caaaacaaagctagagaaac aattccaggg tctcactctg 6240 tcacccaggc tggagtgcag aggcacaatctcggctcact gcaaccttcc tctccctggc 6300 ttaactgatc ctcctacctc agcctcccgagtggctggga ctatactcct gagccagtca 6360 aatatacagc agagtgacta ttctgcagccctaaagcaat gcaacaggga aaagaatcga 6420 acttcttcta tcatccctgt ggaaagatcaagggttggca tttcatccct gagtggagaa 6480 ggcacagact acatcaatgc ctcctatatcatgggctatt accagagcaa tgaattcatc 6540 attacccagc accctctcct tcataccatcaaggatttct ggaggatgat atgggaccat 6600 aatgcccaac tggtggttat gattcctgatggccaaaaca tggcagaaga tgaatttgtt 6660 tactggccaa ataaagatga gcctataaattgtgagagct ttaaggtcac tcttatggct 6720 gaagaacaca aatgtctatc taatgaggaaaaacttataa ttcaggactt tatcttagaa 6780 gctacacagg atgattatgt acttgaagtgaggcactttc agtgtcctaa atggccaaat 6840 ccagatagcc ccattagtaa aacttttgaacttataagtg ttataaaaga agaagctgcc 6900 aatagggatg ggcctatgat tgttcatgatgagcatggag gagtgacggc aggaactttc 6960 tgtgctctga caacccttat gcaccaactagaaaaagaaa attccgtgga tgtttaccag 7020 gtagccaaga tgatcaatct gatgaggccaggagtctttg ctgacattga gcagtatcag 7080 tttctctaca aagtgatcct cagccttgtgagcacaaggc aggaagagaa tccatccacc 7140 tctctggaca gtaatggtgc agcattgcctgatggaaata tagctgagag cttagagtct 7200 ttagtttaac acagaaaggg gtggggggactcacatctga gcattgtttt cctcttccta 7260 aaattaggca ggaaaatcag tctagttctgttatctgttg atttcccatc acctgacagt 7320 aactttcatg acataggatt ctgccgccaaatttatatca ttaacaatgt gtgccttttt 7380 gcaagacttg taatttactt attatgtttgaactaaaatg attgaatttt acagtatttc 7440 taagaatgga attgtggtat ttttttctgtattgatttta acagaaaatt tcaatttata 7500 gaggttagga attccaaact acagaaaatgtttgttttta gtgtcaaatt tttagctgta 7560 tttgtagcaa ttatcaggtt tgctagaaatataactttta atacagtagc ctgtaaataa 7620 aacactcttc catatgatat tcaacattttacaactgcag tattcaccta aagtagaaat 7680 aatctgttac ttattgtaaa tactgccctagtgtctccat ggaccaaatt tatatttata 7740 attgtagatt tttatatttt actactgagtcaagttttct agttctgtgt aattgtttag 7800 tttaatgacg tagttcatta gctggtcttactctaccagt tttctgacat tgtattgtgt 7860 tacctaagtc attaactttg tttcagcatgtaattttaac ttttgtggaa aatagaaata 7920 ccttcatttt gaaagaagtt tttatgagaataacacctta ccaaacattg ttcaaatggt 7980 ttttatccaa ggaattgcaa aaataaatataaatattgcc attaaaaaaa aaaaaaaaaa 8040 aaaaaaaaaa aaaaaaaa 8058 4 2353PRT Homo sapiens gene (1)..(2353) PTP-zeta SM2 23a exon variant 4 MetArg Ile Leu Lys Arg Phe Leu Ala Cys Ile Gln Leu Leu Cys Val 1 5 10 15Cys Arg Leu Asp Trp Ala Asn Gly Tyr Tyr Arg Gln Gln Arg Lys Leu 20 25 30Val Glu Glu Ile Gly Trp Ser Tyr Thr Gly Ala Leu Asn Gln Lys Asn 35 40 45Trp Gly Lys Lys Tyr Pro Thr Cys Asn Ser Pro Lys Gln Ser Pro Ile 50 55 60Asn Ile Asp Glu Asp Leu Thr Gln Val Asn Val Asn Leu Lys Lys Leu 65 70 7580 Lys Phe Gln Gly Trp Asp Lys Thr Ser Leu Glu Asn Thr Phe Ile His 85 9095 Asn Thr Gly Lys Thr Val Glu Ile Asn Leu Thr Asn Asp Tyr Arg Val 100105 110 Ser Gly Gly Val Ser Glu Met Val Phe Lys Ala Ser Lys Ile Thr Phe115 120 125 His Trp Gly Lys Cys Asn Met Ser Ser Asp Gly Ser Glu His SerLeu 130 135 140 Glu Gly Gln Lys Phe Pro Leu Glu Met Gln Ile Tyr Cys PheAsp Ala 145 150 155 160 Asp Arg Phe Ser Ser Phe Glu Glu Ala Val Lys GlyLys Gly Lys Leu 165 170 175 Arg Ala Leu Ser Ile Leu Phe Glu Val Gly ThrGlu Glu Asn Leu Asp 180 185 190 Phe Lys Ala Ile Ile Asp Gly Val Glu SerVal Ser Arg Phe Gly Lys 195 200 205 Gln Ala Ala Leu Asp Pro Phe Ile LeuLeu Asn Leu Leu Pro Asn Ser 210 215 220 Thr Asp Lys Tyr Tyr Ile Tyr AsnGly Ser Leu Thr Ser Pro Pro Cys 225 230 235 240 Thr Asp Thr Val Asp TrpIle Val Phe Lys Asp Thr Val Ser Ile Ser 245 250 255 Glu Ser Gln Leu AlaVal Phe Cys Glu Val Leu Thr Met Gln Gln Ser 260 265 270 Gly Tyr Val MetLeu Met Asp Tyr Leu Gln Asn Asn Phe Arg Glu Gln 275 280 285 Gln Tyr LysPhe Ser Arg Gln Val Phe Ser Ser Tyr Thr Gly Lys Glu 290 295 300 Glu IleHis Glu Ala Val Cys Ser Ser Glu Pro Glu Asn Val Gln Ala 305 310 315 320Asp Pro Glu Asn Tyr Thr Ser Leu Leu Val Thr Trp Glu Arg Pro Arg 325 330335 Val Val Tyr Asp Thr Met Ile Glu Lys Phe Ala Val Leu Tyr Gln Gln 340345 350 Leu Asp Gly Glu Asp Gln Thr Lys His Glu Phe Leu Thr Asp Gly Tyr355 360 365 Gln Asp Leu Gly Ala Ile Leu Asn Asn Leu Leu Pro Asn Met SerTyr 370 375 380 Val Leu Gln Ile Val Ala Ile Cys Thr Asn Gly Leu Tyr GlyLys Tyr 385 390 395 400 Ser Asp Gln Leu Ile Val Asp Met Pro Thr Asp AsnPro Glu Leu Asp 405 410 415 Leu Phe Pro Glu Leu Ile Gly Thr Glu Glu IleIle Lys Glu Glu Glu 420 425 430 Glu Gly Lys Asp Ile Glu Glu Gly Ala IleVal Asn Pro Gly Arg Asp 435 440 445 Ser Ala Thr Asn Gln Ile Arg Lys LysGlu Pro Gln Ile Ser Thr Thr 450 455 460 Thr His Tyr Asn Arg Ile Gly ThrLys Tyr Asn Glu Ala Lys Thr Asn 465 470 475 480 Arg Ser Pro Thr Arg GlySer Glu Phe Ser Gly Lys Gly Asp Val Pro 485 490 495 Asn Thr Ser Leu AsnSer Thr Ser Gln Pro Val Thr Lys Leu Ala Thr 500 505 510 Glu Lys Asp IleSer Leu Thr Ser Gln Thr Val Thr Glu Leu Pro Pro 515 520 525 His Thr ValGlu Gly Thr Ser Ala Ser Leu Asn Asp Gly Ser Lys Thr 530 535 540 Val LeuArg Ser Pro His Met Asn Leu Ser Gly Thr Ala Glu Ser Leu 545 550 555 560Asn Thr Val Ser Ile Thr Glu Tyr Glu Glu Glu Ser Leu Leu Thr Ser 565 570575 Phe Lys Leu Asp Thr Gly Ala Glu Asp Ser Ser Gly Ser Ser Pro Ala 580585 590 Thr Ser Ala Ile Pro Phe Ile Ser Glu Asn Ile Ser Gln Gly Tyr Ile595 600 605 Phe Ser Ser Glu Asn Pro Glu Thr Ile Thr Tyr Asp Val Leu IlePro 610 615 620 Glu Ser Ala Arg Asn Ala Ser Glu Asp Ser Thr Ser Ser GlySer Glu 625 630 635 640 Glu Ser Leu Lys Asp Pro Ser Met Glu Gly Asn ValTrp Phe Pro Ser 645 650 655 Ser Thr Asp Ile Thr Ala Gln Pro Asp Val GlySer Gly Arg Glu Ser 660 665 670 Phe Leu Gln Thr Asn Tyr Thr Glu Ile ArgVal Asp Glu Ser Glu Lys 675 680 685 Thr Thr Lys Ser Phe Ser Ala Gly ProVal Met Ser Gln Gly Pro Ser 690 695 700 Val Thr Asp Leu Glu Met Pro HisTyr Ser Thr Phe Ala Tyr Phe Pro 705 710 715 720 Thr Glu Val Thr Pro HisAla Phe Thr Pro Ser Ser Arg Gln Gln Asp 725 730 735 Leu Val Ser Thr ValAsn Val Val Tyr Ser Gln Thr Thr Gln Pro Val 740 745 750 Tyr Asn Gly GluThr Pro Leu Gln Pro Ser Tyr Ser Ser Glu Val Phe 755 760 765 Pro Leu ValThr Pro Leu Leu Leu Asp Asn Gln Ile Leu Asn Thr Thr 770 775 780 Pro AlaAla Ser Ser Ser Asp Ser Ala Leu His Ala Thr Pro Val Phe 785 790 795 800Pro Ser Val Asp Val Ser Phe Glu Ser Ile Leu Ser Ser Tyr Asp Gly 805 810815 Ala Pro Leu Leu Pro Phe Ser Ser Ala Ser Phe Ser Ser Glu Leu Phe 820825 830 Arg His Leu His Thr Val Ser Gln Ile Leu Pro Gln Val Thr Ser Ala835 840 845 Thr Glu Ser Asp Lys Val Pro Leu His Ala Ser Leu Pro Val AlaGly 850 855 860 Gly Asp Leu Leu Leu Glu Pro Ser Leu Ala Gln Tyr Ser AspVal Leu 865 870 875 880 Ser Thr Thr His Ala Ala Ser Glu Thr Leu Glu PheGly Ser Glu Ser 885 890 895 Gly Val Leu Tyr Lys Thr Leu Met Phe Ser GlnVal Glu Pro Pro Ser 900 905 910 Ser Asp Ala Met Met His Ala Arg Ser SerGly Pro Glu Pro Ser Tyr 915 920 925 Ala Leu Ser Asp Asn Glu Gly Ser GlnHis Ile Phe Thr Val Ser Tyr 930 935 940 Ser Ser Ala Ile Pro Val His AspSer Val Gly Val Thr Tyr Gln Gly 945 950 955 960 Ser Leu Phe Ser Gly ProSer His Ile Pro Ile Pro Lys Ser Ser Leu 965 970 975 Ile Thr Pro Thr AlaSer Leu Leu Gln Pro Thr His Ala Leu Ser Gly 980 985 990 Asp Gly Glu TrpSer Gly Ala Ser Ser Asp Ser Glu Phe Leu Leu Pro 995 1000 1005 Asp ThrAsp Gly Leu Thr Ala Leu Asn Ile Ser Ser Pro Val Ser 1010 1015 1020 ValAla Glu Phe Thr Tyr Thr Thr Ser Val Phe Gly Asp Asp Asn 1025 1030 1035Lys Ala Leu Ser Lys Ser Glu Ile Ile Tyr Gly Asn Glu Thr Glu 1040 10451050 Leu Gln Ile Pro Ser Phe Asn Glu Met Val Tyr Pro Ser Glu Ser 10551060 1065 Thr Val Met Pro Asn Met Tyr Asp Asn Val Asn Lys Leu Asn Ala1070 1075 1080 Ser Leu Gln Glu Thr Ser Val Ser Ile Ser Ser Thr Lys GlyMet 1085 1090 1095 Phe Pro Gly Ser Leu Ala His Thr Thr Thr Lys Val PheAsp His 1100 1105 1110 Glu Ile Ser Gln Val Pro Glu Asn Asn Phe Ser ValGln Pro Thr 1115 1120 1125 His Thr Val Ser Gln Ala Ser Gly Asp Thr SerLeu Lys Pro Val 1130 1135 1140 Leu Ser Ala Asn Ser Glu Pro Ala Ser SerAsp Pro Ala Ser Ser 1145 1150 1155 Glu Met Leu Ser Pro Ser Thr Gln LeuLeu Phe Tyr Glu Thr Ser 1160 1165 1170 Ala Ser Phe Ser Thr Glu Val LeuLeu Gln Pro Ser Phe Gln Ala 1175 1180 1185 Ser Asp Val Asp Thr Leu LeuLys Thr Val Leu Pro Ala Val Pro 1190 1195 1200 Ser Asp Pro Ile Leu ValGlu Thr Pro Lys Val Asp Lys Ile Ser 1205 1210 1215 Ser Thr Met Leu HisLeu Ile Val Ser Asn Ser Ala Ser Ser Glu 1220 1225 1230 Asn Met Leu HisSer Thr Ser Val Pro Val Phe Asp Val Ser Pro 1235 1240 1245 Thr Ser HisMet His Ser Ala Ser Leu Gln Gly Leu Thr Ile Ser 1250 1255 1260 Tyr AlaSer Glu Lys Tyr Glu Pro Val Leu Leu Lys Ser Glu Ser 1265 1270 1275 SerHis Gln Val Val Pro Ser Leu Tyr Ser Asn Asp Glu Leu Phe 1280 1285 1290Gln Thr Ala Asn Leu Glu Ile Asn Gln Ala His Pro Pro Lys Gly 1295 13001305 Arg His Val Phe Ala Thr Pro Val Leu Ser Ile Asp Glu Pro Leu 13101315 1320 Asn Thr Leu Ile Asn Lys Leu Ile His Ser Asp Glu Ile Leu Thr1325 1330 1335 Ser Thr Lys Ser Ser Val Thr Gly Lys Val Phe Ala Gly IlePro 1340 1345 1350 Thr Val Ala Ser Asp Thr Phe Val Ser Thr Asp His SerVal Pro 1355 1360 1365 Ile Gly Asn Gly His Val Ala Ile Thr Ala Val SerPro His Arg 1370 1375 1380 Asp Gly Ser Val Thr Ser Thr Lys Leu Leu PhePro Ser Lys Ala 1385 1390 1395 Thr Ser Glu Leu Ser His Ser Ala Lys SerAsp Ala Gly Leu Val 1400 1405 1410 Gly Gly Gly Glu Asp Gly Asp Thr AspAsp Asp Gly Asp Asp Asp 1415 1420 1425 Asp Asp Arg Asp Ser Asp Gly LeuSer Ile His Lys Cys Met Ser 1430 1435 1440 Cys Ser Ser Tyr Arg Glu SerGln Glu Lys Val Met Asn Asp Ser 1445 1450 1455 Asp Thr His Glu Asn SerLeu Met Asp Gln Asn Asn Pro Ile Ser 1460 1465 1470 Tyr Ser Leu Ser GluAsn Ser Glu Glu Asp Asn Arg Val Thr Ser 1475 1480 1485 Val Ser Ser AspSer Gln Thr Gly Met Asp Arg Ser Pro Gly Lys 1490 1495 1500 Ser Pro SerAla Asn Gly Leu Ser Gln Lys His Asn Asp Gly Lys 1505 1510 1515 Glu GluAsn Asp Ile Gln Thr Gly Ser Ala Leu Leu Pro Leu Ser 1520 1525 1530 ProGlu Ser Lys Ala Trp Ala Val Leu Thr Ser Asp Glu Glu Ser 1535 1540 1545Gly Ser Gly Gln Gly Thr Ser Asp Ser Leu Asn Glu Asn Glu Thr 1550 15551560 Ser Thr Asp Phe Ser Phe Ala Asp Thr Asn Glu Lys Asp Ala Asp 15651570 1575 Gly Ile Leu Ala Ala Gly Asp Ser Glu Ile Thr Pro Gly Phe Pro1580 1585 1590 Gln Ser Pro Thr Ser Ser Val Thr Ser Glu Asn Ser Glu ValPhe 1595 1600 1605 His Val Ser Glu Ala Glu Ala Ser Asn Ser Ser His GluSer Arg 1610 1615 1620 Ile Gly Leu Ala Glu Gly Leu Glu Ser Glu Lys LysAla Val Ile 1625 1630 1635 Pro Leu Val Ile Val Ser Ala Leu Thr Phe IleCys Leu Val Val 1640 1645 1650 Leu Val Gly Ile Leu Ile Tyr Trp Arg LysCys Phe Gln Thr Ala 1655 1660 1665 His Phe Tyr Leu Glu Asp Ser Thr SerPro Arg Val Ile Ser Thr 1670 1675 1680 Pro Pro Thr Pro Ile Phe Pro IleSer Asp Asp Val Gly Ala Ile 1685 1690 1695 Pro Ile Lys His Phe Pro LysHis Val Ala Asp Leu His Ala Ser 1700 1705 1710 Ser Gly Phe Thr Glu GluPhe Glu Thr Leu Lys Glu Phe Tyr Gln 1715 1720 1725 Glu Val Gln Ser CysThr Val Asp Leu Gly Ile Thr Ala Asp Ser 1730 1735 1740 Ser Asn His ProAsp Asn Lys His Lys Asn Arg Tyr Ile Asn Ile 1745 1750 1755 Val Ala TyrAsp His Ser Arg Val Lys Leu Ala Gln Leu Ala Glu 1760 1765 1770 Lys AspGly Lys Leu Thr Asp Tyr Ile Asn Ala Asn Tyr Val Asp 1775 1780 1785 GlyTyr Asn Arg Pro Lys Ala Tyr Ile Ala Ala Gln Gly Pro Leu 1790 1795 1800Lys Ser Thr Ala Glu Asp Phe Trp Arg Met Ile Trp Glu His Asn 1805 18101815 Val Glu Val Ile Val Met Ile Thr Asn Leu Val Glu Lys Gly Arg 18201825 1830 Arg Lys Cys Asp Gln Tyr Trp Pro Ala Asp Gly Ser Glu Glu Tyr1835 1840 1845 Gly Asn Phe Leu Val Thr Gln Lys Ser Val Gln Val Leu AlaTyr 1850 1855 1860 Tyr Thr Val Arg Asn Phe Thr Leu Arg Asn Thr Lys IleLys Lys 1865 1870 1875 Gly Ser Gln Lys Gly Arg Pro Ser Gly Arg Val ValThr Gln Tyr 1880 1885 1890 His Tyr Thr Gln Trp Pro Asp Met Gly Val ProGlu Tyr Ser Leu 1895 1900 1905 Pro Val Leu Thr Phe Val Arg Lys Ala AlaTyr Ala Lys Arg His 1910 1915 1920 Ala Val Gly Pro Val Val Val His CysSer Ala Gly Val Gly Arg 1925 1930 1935 Thr Gly Thr Tyr Ile Val Leu AspSer Met Leu Gln Gln Ile Gln 1940 1945 1950 His Glu Gly Thr Val Asn IlePhe Gly Phe Leu Lys His Ile Arg 1955 1960 1965 Ser Gln Arg Asn Tyr LeuVal Gln Thr Glu Glu Gln Tyr Val Phe 1970 1975 1980 Ile His Asp Thr LeuVal Glu Ala Ile Leu Ser Lys Glu Thr Glu 1985 1990 1995 Val Leu Asp SerHis Ile His Ala Tyr Val Asn Ala Leu Leu Ile 2000 2005 2010 Pro Gly ProAla Gly Lys Thr Lys Leu Glu Lys Gln Phe Gln Gly 2015 2020 2025 Leu ThrLeu Ser Pro Arg Leu Glu Cys Arg Gly Thr Ile Ser Ala 2030 2035 2040 HisCys Asn Leu Pro Leu Pro Gly Leu Thr Asp Pro Pro Thr Ser 2045 2050 2055Ala Ser Arg Val Ala Gly Thr Ile Leu Leu Ser Gln Ser Asn Ile 2060 20652070 Gln Gln Ser Asp Tyr Ser Ala Ala Leu Lys Gln Cys Asn Arg Glu 20752080 2085 Lys Asn Arg Thr Ser Ser Ile Ile Pro Val Glu Arg Ser Arg Val2090 2095 2100 Gly Ile Ser Ser Leu Ser Gly Glu Gly Thr Asp Tyr Ile AsnAla 2105 2110 2115 Ser Tyr Ile Met Gly Tyr Tyr Gln Ser Asn Glu Phe IleIle Thr 2120 2125 2130 Gln His Pro Leu Leu His Thr Ile Lys Asp Phe TrpArg Met Ile 2135 2140 2145 Trp Asp His Asn Ala Gln Leu Val Val Met IlePro Asp Gly Gln 2150 2155 2160 Asn Met Ala Glu Asp Glu Phe Val Tyr TrpPro Asn Lys Asp Glu 2165 2170 2175 Pro Ile Asn Cys Glu Ser Phe Lys ValThr Leu Met Ala Glu Glu 2180 2185 2190 His Lys Cys Leu Ser Asn Glu GluLys Leu Ile Ile Gln Asp Phe 2195 2200 2205 Ile Leu Glu Ala Thr Gln AspAsp Tyr Val Leu Glu Val Arg His 2210 2215 2220 Phe Gln Cys Pro Lys TrpPro Asn Pro Asp Ser Pro Ile Ser Lys 2225 2230 2235 Thr Phe Glu Leu IleSer Val Ile Lys Glu Glu Ala Ala Asn Arg 2240 2245 2250 Asp Gly Pro MetIle Val His Asp Glu His Gly Gly Val Thr Ala 2255 2260 2265 Gly Thr PheCys Ala Leu Thr Thr Leu Met His Gln Leu Glu Lys 2270 2275 2280 Glu AsnSer Val Asp Val Tyr Gln Val Ala Lys Met Ile Asn Leu 2285 2290 2295 MetArg Pro Gly Val Phe Ala Asp Ile Glu Gln Tyr Gln Phe Leu 2300 2305 2310Tyr Lys Val Ile Leu Ser Leu Val Ser Thr Arg Gln Glu Glu Asn 2315 23202325 Pro Ser Thr Ser Leu Asp Ser Asn Gly Ala Ala Leu Pro Asp Gly 23302335 2340 Asn Ile Ala Glu Ser Leu Glu Ser Leu Val 2345 2350 5 7941 DNAHomo sapiens CDS (148)..(7092) 5 cacacatacg cacgcacgat ctcacttcgatctatacact ggaggattaa aacaaacaaa 60 caaaaaaaac atttccttcg ctccccctccctctccactc tgagaagcag aggagccgca 120 cggcgagggg ccgcagaccg tctggaa atgcga atc cta aag cgt ttc ctc gct 174 Met Arg Ile Leu Lys Arg Phe Leu Ala1 5 tgc att cag ctc ctc tgt gtt tgc cgc ctg gat tgg gct aat gga tac 222Cys Ile Gln Leu Leu Cys Val Cys Arg Leu Asp Trp Ala Asn Gly Tyr 10 15 2025 tac aga caa cag aga aaa ctt gtt gaa gag att ggc tgg tcc tat aca 270Tyr Arg Gln Gln Arg Lys Leu Val Glu Glu Ile Gly Trp Ser Tyr Thr 30 35 40gga gca ctg aat caa aaa aat tgg gga aag aaa tat cca aca tgt aat 318 GlyAla Leu Asn Gln Lys Asn Trp Gly Lys Lys Tyr Pro Thr Cys Asn 45 50 55 agccca aaa caa tct cct atc aat att gat gaa gat ctt aca caa gta 366 Ser ProLys Gln Ser Pro Ile Asn Ile Asp Glu Asp Leu Thr Gln Val 60 65 70 aat gtgaat ctt aag aaa ctt aaa ttt cag ggt tgg gat aaa aca tca 414 Asn Val AsnLeu Lys Lys Leu Lys Phe Gln Gly Trp Asp Lys Thr Ser 75 80 85 ttg gaa aacaca ttc att cat aac act ggg aaa aca gtg gaa att aat 462 Leu Glu Asn ThrPhe Ile His Asn Thr Gly Lys Thr Val Glu Ile Asn 90 95 100 105 ctc actaat gac tac cgt gtc agc gga gga gtt tca gaa atg gtg ttt 510 Leu Thr AsnAsp Tyr Arg Val Ser Gly Gly Val Ser Glu Met Val Phe 110 115 120 aaa gcaagc aag ata act ttt cac tgg gga aaa tgc aat atg tca tct 558 Lys Ala SerLys Ile Thr Phe His Trp Gly Lys Cys Asn Met Ser Ser 125 130 135 gat ggatca gag cat agt tta gaa gga caa aaa ttt cca ctt gag atg 606 Asp Gly SerGlu His Ser Leu Glu Gly Gln Lys Phe Pro Leu Glu Met 140 145 150 caa atctac tgc ttt gat gcg gac cga ttt tca agt ttt gag gaa gca 654 Gln Ile TyrCys Phe Asp Ala Asp Arg Phe Ser Ser Phe Glu Glu Ala 155 160 165 gtc aaagga aaa ggg aag tta aga gct tta tcc att ttg ttt gag gtt 702 Val Lys GlyLys Gly Lys Leu Arg Ala Leu Ser Ile Leu Phe Glu Val 170 175 180 185 gggaca gaa gaa aat ttg gat ttc aaa gcg att att gat gga gtc gaa 750 Gly ThrGlu Glu Asn Leu Asp Phe Lys Ala Ile Ile Asp Gly Val Glu 190 195 200 agtgtt agt cgt ttt ggg aag cag gct gct tta gat cca ttc ata ctg 798 Ser ValSer Arg Phe Gly Lys Gln Ala Ala Leu Asp Pro Phe Ile Leu 205 210 215 ttgaac ctt ctg cca aac tca act gac aag tat tac att tac aat ggc 846 Leu AsnLeu Leu Pro Asn Ser Thr Asp Lys Tyr Tyr Ile Tyr Asn Gly 220 225 230 tcattg aca tct cct ccc tgc aca gac aca gtt gac tgg att gtt ttt 894 Ser LeuThr Ser Pro Pro Cys Thr Asp Thr Val Asp Trp Ile Val Phe 235 240 245 aaagat aca gtt agc atc tct gaa agc cag ttg gct gtt ttt tgt gaa 942 Lys AspThr Val Ser Ile Ser Glu Ser Gln Leu Ala Val Phe Cys Glu 250 255 260 265gtt ctt aca atg caa caa tct ggt tat gtc atg ctg atg gac tac tta 990 ValLeu Thr Met Gln Gln Ser Gly Tyr Val Met Leu Met Asp Tyr Leu 270 275 280caa aac aat ttt cga gag caa cag tac aag ttc tct aga cag gtg ttt 1038 GlnAsn Asn Phe Arg Glu Gln Gln Tyr Lys Phe Ser Arg Gln Val Phe 285 290 295tcc tca tac act gga aag gaa gag att cat gaa gca gtt tgt agt tca 1086 SerSer Tyr Thr Gly Lys Glu Glu Ile His Glu Ala Val Cys Ser Ser 300 305 310gaa cca gaa aat gtt cag gct gac cca gag aat tat acc agc ctt ctt 1134 GluPro Glu Asn Val Gln Ala Asp Pro Glu Asn Tyr Thr Ser Leu Leu 315 320 325gtt aca tgg gaa aga cct cga gtc gtt tat gat acc atg att gag aag 1182 ValThr Trp Glu Arg Pro Arg Val Val Tyr Asp Thr Met Ile Glu Lys 330 335 340345 ttt gca gtt ttg tac cag cag ttg gat gga gag gac caa acc aag cat 1230Phe Ala Val Leu Tyr Gln Gln Leu Asp Gly Glu Asp Gln Thr Lys His 350 355360 gaa ttt ttg aca gat ggc tat caa gac ttg ggt gct att ctc aat aat 1278Glu Phe Leu Thr Asp Gly Tyr Gln Asp Leu Gly Ala Ile Leu Asn Asn 365 370375 ttg cta ccc aat atg agt tat gtt ctt cag ata gta gcc ata tgc act 1326Leu Leu Pro Asn Met Ser Tyr Val Leu Gln Ile Val Ala Ile Cys Thr 380 385390 aat ggc tta tat gga aaa tac agc gac caa ctg att gtc gac atg cct 1374Asn Gly Leu Tyr Gly Lys Tyr Ser Asp Gln Leu Ile Val Asp Met Pro 395 400405 act gat aat cct gaa ctt gat ctt ttc cct gaa tta att gga act gaa 1422Thr Asp Asn Pro Glu Leu Asp Leu Phe Pro Glu Leu Ile Gly Thr Glu 410 415420 425 gaa ata atc aag gag gag gaa gag gga aaa gac att gaa gaa ggc gct1470 Glu Ile Ile Lys Glu Glu Glu Glu Gly Lys Asp Ile Glu Glu Gly Ala 430435 440 att gtg aat cct ggt aga gac agt gct aca aac caa atc agg aaa aag1518 Ile Val Asn Pro Gly Arg Asp Ser Ala Thr Asn Gln Ile Arg Lys Lys 445450 455 gaa ccc cag att tct acc aca aca cac tac aat cgc ata ggg acg aaa1566 Glu Pro Gln Ile Ser Thr Thr Thr His Tyr Asn Arg Ile Gly Thr Lys 460465 470 tac aat gaa gcc aag act aac cga tcc cca aca aga gga agt gaa ttc1614 Tyr Asn Glu Ala Lys Thr Asn Arg Ser Pro Thr Arg Gly Ser Glu Phe 475480 485 tct gga aag ggt gat gtt ccc aat aca tct tta aat tcc act tcc caa1662 Ser Gly Lys Gly Asp Val Pro Asn Thr Ser Leu Asn Ser Thr Ser Gln 490495 500 505 cca gtc act aaa tta gcc aca gaa aaa gat att tcc ttg act tctcag 1710 Pro Val Thr Lys Leu Ala Thr Glu Lys Asp Ile Ser Leu Thr Ser Gln510 515 520 act gtg act gaa ctg cca cct cac act gtg gaa ggt act tca gcctct 1758 Thr Val Thr Glu Leu Pro Pro His Thr Val Glu Gly Thr Ser Ala Ser525 530 535 tta aat gat ggc tct aaa act gtt ctt aga tct cca cat atg aacttg 1806 Leu Asn Asp Gly Ser Lys Thr Val Leu Arg Ser Pro His Met Asn Leu540 545 550 tcg ggg act gca gaa tcc tta aat aca gtt tct ata aca gaa tatgag 1854 Ser Gly Thr Ala Glu Ser Leu Asn Thr Val Ser Ile Thr Glu Tyr Glu555 560 565 gag gag agt tta ttg acc agt ttc aag ctt gat act gga gct gaagat 1902 Glu Glu Ser Leu Leu Thr Ser Phe Lys Leu Asp Thr Gly Ala Glu Asp570 575 580 585 tct tca ggc tcc agt ccc gca act tct gct atc cca ttc atctct gag 1950 Ser Ser Gly Ser Ser Pro Ala Thr Ser Ala Ile Pro Phe Ile SerGlu 590 595 600 aac ata tcc caa ggg tat ata ttt tcc tcc gaa aac cca gagaca ata 1998 Asn Ile Ser Gln Gly Tyr Ile Phe Ser Ser Glu Asn Pro Glu ThrIle 605 610 615 aca tat gat gtc ctt ata cca gaa tct gct aga aat gct tccgaa gat 2046 Thr Tyr Asp Val Leu Ile Pro Glu Ser Ala Arg Asn Ala Ser GluAsp 620 625 630 tca act tca tca ggt tca gaa gaa tca cta aag gat cct tctatg gag 2094 Ser Thr Ser Ser Gly Ser Glu Glu Ser Leu Lys Asp Pro Ser MetGlu 635 640 645 gga aat gtg tgg ttt cct agc tct aca gac ata aca gca cagccc gat 2142 Gly Asn Val Trp Phe Pro Ser Ser Thr Asp Ile Thr Ala Gln ProAsp 650 655 660 665 gtt gga tca ggc aga gag agc ttt ctc cag act aat tacact gag ata 2190 Val Gly Ser Gly Arg Glu Ser Phe Leu Gln Thr Asn Tyr ThrGlu Ile 670 675 680 cgt gtt gat gaa tct gag aag aca acc aag tcc ttt tctgca ggc cca 2238 Arg Val Asp Glu Ser Glu Lys Thr Thr Lys Ser Phe Ser AlaGly Pro 685 690 695 gtg atg tca cag ggt ccc tca gtt aca gat ctg gaa atgcca cat tat 2286 Val Met Ser Gln Gly Pro Ser Val Thr Asp Leu Glu Met ProHis Tyr 700 705 710 tct acc ttt gcc tac ttc cca act gag gta aca cct catgct ttt acc 2334 Ser Thr Phe Ala Tyr Phe Pro Thr Glu Val Thr Pro His AlaPhe Thr 715 720 725 cca tcc tcc aga caa cag gat ttg gtc tcc acg gtc aacgtg gta tac 2382 Pro Ser Ser Arg Gln Gln Asp Leu Val Ser Thr Val Asn ValVal Tyr 730 735 740 745 tcg cag aca acc caa ccg gta tac aat ggt gag acacct ctt caa cct 2430 Ser Gln Thr Thr Gln Pro Val Tyr Asn Gly Glu Thr ProLeu Gln Pro 750 755 760 tcc tac agt agt gaa gtc ttt cct cta gtc acc cctttg ttg ctt gac 2478 Ser Tyr Ser Ser Glu Val Phe Pro Leu Val Thr Pro LeuLeu Leu Asp 765 770 775 aat cag atc ctc aac act acc cct gct gct tca agtagt gat tcg gcc 2526 Asn Gln Ile Leu Asn Thr Thr Pro Ala Ala Ser Ser SerAsp Ser Ala 780 785 790 ttg cat gct acg cct gta ttt ccc agt gtc gat gtgtca ttt gaa tcc 2574 Leu His Ala Thr Pro Val Phe Pro Ser Val Asp Val SerPhe Glu Ser 795 800 805 atc ctg tct tcc tat gat ggt gca cct ttg ctt ccattt tcc tct gct 2622 Ile Leu Ser Ser Tyr Asp Gly Ala Pro Leu Leu Pro PheSer Ser Ala 810 815 820 825 tcc ttc agt agt gaa ttg ttt cgc cat ctg cataca gtt tct caa atc 2670 Ser Phe Ser Ser Glu Leu Phe Arg His Leu His ThrVal Ser Gln Ile 830 835 840 ctt cca caa gtt act tca gct acc gag agt gataag gtg ccc ttg cat 2718 Leu Pro Gln Val Thr Ser Ala Thr Glu Ser Asp LysVal Pro Leu His 845 850 855 gct tct ctg cca gtg gct ggg ggt gat ttg ctatta gag ccc agc ctt 2766 Ala Ser Leu Pro Val Ala Gly Gly Asp Leu Leu LeuGlu Pro Ser Leu 860 865 870 gct cag tat tct gat gtg ctg tcc act act catgct gct tca gag acg 2814 Ala Gln Tyr Ser Asp Val Leu Ser Thr Thr His AlaAla Ser Glu Thr 875 880 885 ctg gaa ttt ggt agt gaa tct ggt gtt ctt tataaa acg ctt atg ttt 2862 Leu Glu Phe Gly Ser Glu Ser Gly Val Leu Tyr LysThr Leu Met Phe 890 895 900 905 tct caa gtt gaa cca ccc agc agt gat gccatg atg cat gca cgt tct 2910 Ser Gln Val Glu Pro Pro Ser Ser Asp Ala MetMet His Ala Arg Ser 910 915 920 tca ggg cct gaa cct tct tat gcc ttg tctgat aat gag ggc tcc caa 2958 Ser Gly Pro Glu Pro Ser Tyr Ala Leu Ser AspAsn Glu Gly Ser Gln 925 930 935 cac atc ttc act gtt tct tac agt tct gcaata cct gtg cat gat tct 3006 His Ile Phe Thr Val Ser Tyr Ser Ser Ala IlePro Val His Asp Ser 940 945 950 gtg ggt gta act tat cag ggt tcc tta tttagc ggc cct agc cat ata 3054 Val Gly Val Thr Tyr Gln Gly Ser Leu Phe SerGly Pro Ser His Ile 955 960 965 cca ata cct aag tct tcg tta ata acc ccaact gca tca tta ctg cag 3102 Pro Ile Pro Lys Ser Ser Leu Ile Thr Pro ThrAla Ser Leu Leu Gln 970 975 980 985 cct act cat gcc ctc tct ggt gat ggggaa tgg tct gga gcc tct tct 3150 Pro Thr His Ala Leu Ser Gly Asp Gly GluTrp Ser Gly Ala Ser Ser 990 995 1000 gat agt gaa ttt ctt tta cct gac acagat ggg ctg aca gcc ctt 3195 Asp Ser Glu Phe Leu Leu Pro Asp Thr Asp GlyLeu Thr Ala Leu 1005 1010 1015 aac att tct tca cct gtt tct gta gct gaattt aca tat aca aca 3240 Asn Ile Ser Ser Pro Val Ser Val Ala Glu Phe ThrTyr Thr Thr 1020 1025 1030 tct gtg ttt ggt gat gat aat aag gcg ctt tctaaa agt gaa ata 3285 Ser Val Phe Gly Asp Asp Asn Lys Ala Leu Ser Lys SerGlu Ile 1035 1040 1045 ata tat gga aat gag act gaa ctg caa att cct tctttc aat gag 3330 Ile Tyr Gly Asn Glu Thr Glu Leu Gln Ile Pro Ser Phe AsnGlu 1050 1055 1060 atg gtt tac cct tct gaa agc aca gtc atg ccc aac atgtat gat 3375 Met Val Tyr Pro Ser Glu Ser Thr Val Met Pro Asn Met Tyr Asp1065 1070 1075 aat gta aat aag ttg aat gcg tct tta caa gaa acc tct gtttcc 3420 Asn Val Asn Lys Leu Asn Ala Ser Leu Gln Glu Thr Ser Val Ser1080 1085 1090 att tct agc acc aag ggc atg ttt cca ggg tcc ctt gct catacc 3465 Ile Ser Ser Thr Lys Gly Met Phe Pro Gly Ser Leu Ala His Thr1095 1100 1105 acc act aag gtt ttt gat cat gag att agt caa gtt cca gaaaat 3510 Thr Thr Lys Val Phe Asp His Glu Ile Ser Gln Val Pro Glu Asn1110 1115 1120 aac ttt tca gtt caa cct aca cat act gtc tct caa gca tctggt 3555 Asn Phe Ser Val Gln Pro Thr His Thr Val Ser Gln Ala Ser Gly1125 1130 1135 gac act tcg ctt aaa cct gtg ctt agt gca aac tca gag ccagca 3600 Asp Thr Ser Leu Lys Pro Val Leu Ser Ala Asn Ser Glu Pro Ala1140 1145 1150 tcc tct gac cct gct tct agt gaa atg tta tct cct tca actcag 3645 Ser Ser Asp Pro Ala Ser Ser Glu Met Leu Ser Pro Ser Thr Gln1155 1160 1165 ctc tta ttt tat gag acc tca gct tct ttt agt act gaa gtattg 3690 Leu Leu Phe Tyr Glu Thr Ser Ala Ser Phe Ser Thr Glu Val Leu1170 1175 1180 cta caa cct tcc ttt cag gct tct gat gtt gac acc ttg cttaaa 3735 Leu Gln Pro Ser Phe Gln Ala Ser Asp Val Asp Thr Leu Leu Lys1185 1190 1195 act gtt ctt cca gct gtg ccc agt gat cca ata ttg gtt gaaacc 3780 Thr Val Leu Pro Ala Val Pro Ser Asp Pro Ile Leu Val Glu Thr1200 1205 1210 ccc aaa gtt gat aaa att agt tct aca atg ttg cat ctc attgta 3825 Pro Lys Val Asp Lys Ile Ser Ser Thr Met Leu His Leu Ile Val1215 1220 1225 tca aat tct gct tca agt gaa aac atg ctg cac tct aca tctgta 3870 Ser Asn Ser Ala Ser Ser Glu Asn Met Leu His Ser Thr Ser Val1230 1235 1240 cca gtt ttt gat gtg tcg cct act tct cat atg cac tct gcttca 3915 Pro Val Phe Asp Val Ser Pro Thr Ser His Met His Ser Ala Ser1245 1250 1255 ctt caa ggt ttg acc att tcc tat gca agt gag aaa tat gaacca 3960 Leu Gln Gly Leu Thr Ile Ser Tyr Ala Ser Glu Lys Tyr Glu Pro1260 1265 1270 gtt ttg tta aaa agt gaa agt tcc cac caa gtg gta cct tctttg 4005 Val Leu Leu Lys Ser Glu Ser Ser His Gln Val Val Pro Ser Leu1275 1280 1285 tac agt aat gat gag ttg ttc caa acg gcc aat ttg gag attaac 4050 Tyr Ser Asn Asp Glu Leu Phe Gln Thr Ala Asn Leu Glu Ile Asn1290 1295 1300 cag gcc cat ccc cca aaa gga agg cat gta ttt gct aca cctgtt 4095 Gln Ala His Pro Pro Lys Gly Arg His Val Phe Ala Thr Pro Val1305 1310 1315 tta tca att gat gaa cca tta aat aca cta ata aat aag cttata 4140 Leu Ser Ile Asp Glu Pro Leu Asn Thr Leu Ile Asn Lys Leu Ile1320 1325 1330 cat tcc gat gaa att tta acc tcc acc aaa agt tct gtt actggt 4185 His Ser Asp Glu Ile Leu Thr Ser Thr Lys Ser Ser Val Thr Gly1335 1340 1345 aag gta ttt gct ggt att cca aca gtt gct tct gat aca tttgta 4230 Lys Val Phe Ala Gly Ile Pro Thr Val Ala Ser Asp Thr Phe Val1350 1355 1360 tct act gat cat tct gtt cct ata gga aat ggg cat gtt gccatt 4275 Ser Thr Asp His Ser Val Pro Ile Gly Asn Gly His Val Ala Ile1365 1370 1375 aca gct gtt tct ccc cac aga gat ggt tct gta acc tca acaaag 4320 Thr Ala Val Ser Pro His Arg Asp Gly Ser Val Thr Ser Thr Lys1380 1385 1390 ttg ctg ttt cct tct aag gca act tct gag ctg agt cat agtgcc 4365 Leu Leu Phe Pro Ser Lys Ala Thr Ser Glu Leu Ser His Ser Ala1395 1400 1405 aaa tct gat gcc ggt tta gtg ggt ggt ggt gaa gat ggt gacact 4410 Lys Ser Asp Ala Gly Leu Val Gly Gly Gly Glu Asp Gly Asp Thr1410 1415 1420 gat gat gat ggt gat gat gat gat gac aga gat agt gat ggctta 4455 Asp Asp Asp Gly Asp Asp Asp Asp Asp Arg Asp Ser Asp Gly Leu1425 1430 1435 tcc att cat aag tgt atg tca tgc tca tcc tat aga gaa tcacag 4500 Ser Ile His Lys Cys Met Ser Cys Ser Ser Tyr Arg Glu Ser Gln1440 1445 1450 gaa aag gta atg aat gat tca gac acc cac gaa aac agt cttatg 4545 Glu Lys Val Met Asn Asp Ser Asp Thr His Glu Asn Ser Leu Met1455 1460 1465 gat cag aat aat cca atc tca tac tca cta tct gag aat tctgaa 4590 Asp Gln Asn Asn Pro Ile Ser Tyr Ser Leu Ser Glu Asn Ser Glu1470 1475 1480 gaa gat aat aga gtc aca agt gta tcc tca gac agt caa actggt 4635 Glu Asp Asn Arg Val Thr Ser Val Ser Ser Asp Ser Gln Thr Gly1485 1490 1495 atg gac aga agt cct ggt aaa tca cca tca gca aat ggg ctatcc 4680 Met Asp Arg Ser Pro Gly Lys Ser Pro Ser Ala Asn Gly Leu Ser1500 1505 1510 caa aag cac aat gat gga aaa gag gaa aat gac att cag actggt 4725 Gln Lys His Asn Asp Gly Lys Glu Glu Asn Asp Ile Gln Thr Gly1515 1520 1525 agt gct ctg ctt cct ctc agc cct gaa tct aaa gca tgg gcagtt 4770 Ser Ala Leu Leu Pro Leu Ser Pro Glu Ser Lys Ala Trp Ala Val1530 1535 1540 ctg aca agt gat gaa gaa agt gga tca ggg caa ggt acc tcagat 4815 Leu Thr Ser Asp Glu Glu Ser Gly Ser Gly Gln Gly Thr Ser Asp1545 1550 1555 agc ctt aat gag aat gag act tcc aca gat ttc agt ttt gcagac 4860 Ser Leu Asn Glu Asn Glu Thr Ser Thr Asp Phe Ser Phe Ala Asp1560 1565 1570 act aat gaa aaa gat gct gat ggg atc ctg gca gca ggt gactca 4905 Thr Asn Glu Lys Asp Ala Asp Gly Ile Leu Ala Ala Gly Asp Ser1575 1580 1585 gaa ata act cct gga ttc cca cag tcc cca aca tca tct gttact 4950 Glu Ile Thr Pro Gly Phe Pro Gln Ser Pro Thr Ser Ser Val Thr1590 1595 1600 agc gag aac tca gaa gtg ttc cac gtt tca gag gca gag gccagt 4995 Ser Glu Asn Ser Glu Val Phe His Val Ser Glu Ala Glu Ala Ser1605 1610 1615 aat agt agc cat gag tct cgt att ggt cta gct gag ggg ttggaa 5040 Asn Ser Ser His Glu Ser Arg Ile Gly Leu Ala Glu Gly Leu Glu1620 1625 1630 tcc gag aag aag gca gtt ata ccc ctt gtg atc gtg tca gccctg 5085 Ser Glu Lys Lys Ala Val Ile Pro Leu Val Ile Val Ser Ala Leu1635 1640 1645 act ttt atc tgt cta gtg gtt ctt gtg ggt att ctc atc tactgg 5130 Thr Phe Ile Cys Leu Val Val Leu Val Gly Ile Leu Ile Tyr Trp1650 1655 1660 agg aaa tgc ttc cag act gca cac ttt tac tta gag gac agtaca 5175 Arg Lys Cys Phe Gln Thr Ala His Phe Tyr Leu Glu Asp Ser Thr1665 1670 1675 tcc cct aga gtt ata tcc aca cct cca aca cct atc ttt ccaatt 5220 Ser Pro Arg Val Ile Ser Thr Pro Pro Thr Pro Ile Phe Pro Ile1680 1685 1690 tca gat gat gtc gga gca att cca ata aag cac ttt cca aagcat 5265 Ser Asp Asp Val Gly Ala Ile Pro Ile Lys His Phe Pro Lys His1695 1700 1705 gtt gca gat tta cat gca agt agt ggg ttt act gaa gaa tttgag 5310 Val Ala Asp Leu His Ala Ser Ser Gly Phe Thr Glu Glu Phe Glu1710 1715 1720 aca ctg aaa gag ttt tac cag gaa gtg cag agc tgt act gttgac 5355 Thr Leu Lys Glu Phe Tyr Gln Glu Val Gln Ser Cys Thr Val Asp1725 1730 1735 tta ggt att aca gca gac agc tcc aac cac cca gac aac aagcac 5400 Leu Gly Ile Thr Ala Asp Ser Ser Asn His Pro Asp Asn Lys His1740 1745 1750 aag aat cga tac ata aat atc gtt gcc tat gat cat agc agggtt 5445 Lys Asn Arg Tyr Ile Asn Ile Val Ala Tyr Asp His Ser Arg Val1755 1760 1765 aag cta gca cag ctt gct gaa aag gat ggc aaa ctg act gattat 5490 Lys Leu Ala Gln Leu Ala Glu Lys Asp Gly Lys Leu Thr Asp Tyr1770 1775 1780 atc aat gcc aat tat gtt gat ggc tac aac aga cca aaa gcttat 5535 Ile Asn Ala Asn Tyr Val Asp Gly Tyr Asn Arg Pro Lys Ala Tyr1785 1790 1795 att gct gcc caa ggc cca ctg aaa tcc aca gct gaa gat ttctgg 5580 Ile Ala Ala Gln Gly Pro Leu Lys Ser Thr Ala Glu Asp Phe Trp1800 1805 1810 aga atg ata tgg gaa cat aat gtg gaa gtt att gtc atg ataaca 5625 Arg Met Ile Trp Glu His Asn Val Glu Val Ile Val Met Ile Thr1815 1820 1825 aac ctc gtg gag aaa gga agg aga aaa tgt gat cag tac tggcct 5670 Asn Leu Val Glu Lys Gly Arg Arg Lys Cys Asp Gln Tyr Trp Pro1830 1835 1840 gcc gat ggg agt gag gag tac ggg aac ttt ctg gtc act cagaag 5715 Ala Asp Gly Ser Glu Glu Tyr Gly Asn Phe Leu Val Thr Gln Lys1845 1850 1855 agt gtg caa gtg ctt gcc tat tat act gtg agg aat ttt actcta 5760 Ser Val Gln Val Leu Ala Tyr Tyr Thr Val Arg Asn Phe Thr Leu1860 1865 1870 aga aac aca aaa ata aaa aag ggc tcc cag aaa gga aga cccagt 5805 Arg Asn Thr Lys Ile Lys Lys Gly Ser Gln Lys Gly Arg Pro Ser1875 1880 1885 gga cgt gtg gtc aca cag tat cac tac acg cag tgg cct gacatg 5850 Gly Arg Val Val Thr Gln Tyr His Tyr Thr Gln Trp Pro Asp Met1890 1895 1900 gga gta cca gag tac tcc ctg cca gtg ctg acc ttt gtg agaaag 5895 Gly Val Pro Glu Tyr Ser Leu Pro Val Leu Thr Phe Val Arg Lys1905 1910 1915 gca gcc tat gcc aag cgc cat gca gtg ggg cct gtt gtc gtccac 5940 Ala Ala Tyr Ala Lys Arg His Ala Val Gly Pro Val Val Val His1920 1925 1930 tgc agt gct gga gtt gga aga aca ggc aca tat att gtg ctagac 5985 Cys Ser Ala Gly Val Gly Arg Thr Gly Thr Tyr Ile Val Leu Asp1935 1940 1945 agt atg ttg cag cag att caa cac gaa gga act gtc aac atattt 6030 Ser Met Leu Gln Gln Ile Gln His Glu Gly Thr Val Asn Ile Phe1950 1955 1960 ggc ttc tta aaa cac atc cgt tca caa aga aat tat ttg gtacaa 6075 Gly Phe Leu Lys His Ile Arg Ser Gln Arg Asn Tyr Leu Val Gln1965 1970 1975 act gag gag caa tat gtc ttc att cat gat aca ctg gtt gaggcc 6120 Thr Glu Glu Gln Tyr Val Phe Ile His Asp Thr Leu Val Glu Ala1980 1985 1990 ata ctt agt aaa gaa act gag gtg ctg gac agt cat att catgcc 6165 Ile Leu Ser Lys Glu Thr Glu Val Leu Asp Ser His Ile His Ala1995 2000 2005 tat gtt aat gca ctc ctc att cct gga cca gca ggc aaa acaaag 6210 Tyr Val Asn Ala Leu Leu Ile Pro Gly Pro Ala Gly Lys Thr Lys2010 2015 2020 cta gag aaa caa ttc cag ctc ctg agc cag tca aat ata cagcag 6255 Leu Glu Lys Gln Phe Gln Leu Leu Ser Gln Ser Asn Ile Gln Gln2025 2030 2035 agt gac tat tct gca gcc cta aag caa tgc aac agg gaa aagaat 6300 Ser Asp Tyr Ser Ala Ala Leu Lys Gln Cys Asn Arg Glu Lys Asn2040 2045 2050 cga act tct tct atc atc cct gtg gaa aga tca agg gtt ggcatt 6345 Arg Thr Ser Ser Ile Ile Pro Val Glu Arg Ser Arg Val Gly Ile2055 2060 2065 tca tcc ctg agt gga gaa ggc aca gac tac atc aat gcc tcctat 6390 Ser Ser Leu Ser Gly Glu Gly Thr Asp Tyr Ile Asn Ala Ser Tyr2070 2075 2080 atc atg ggc tat tac cag agc aat gaa ttc atc att acc cagcac 6435 Ile Met Gly Tyr Tyr Gln Ser Asn Glu Phe Ile Ile Thr Gln His2085 2090 2095 cct ctc ctt cat acc atc aag gat ttc tgg agg atg ata tgggac 6480 Pro Leu Leu His Thr Ile Lys Asp Phe Trp Arg Met Ile Trp Asp2100 2105 2110 cat aat gcc caa ctg gtg gtt atg att cct gat ggc caa aacatg 6525 His Asn Ala Gln Leu Val Val Met Ile Pro Asp Gly Gln Asn Met2115 2120 2125 gca gaa gat gaa ttt gtt tac tgg cca aat aaa gat gag cctata 6570 Ala Glu Asp Glu Phe Val Tyr Trp Pro Asn Lys Asp Glu Pro Ile2130 2135 2140 aat tgt gag agc ttt aag gtc act ctt atg gct gaa gaa cacaaa 6615 Asn Cys Glu Ser Phe Lys Val Thr Leu Met Ala Glu Glu His Lys2145 2150 2155 tgt cta tct aat gag gaa aaa ctt ata att cag gac ttt atctta 6660 Cys Leu Ser Asn Glu Glu Lys Leu Ile Ile Gln Asp Phe Ile Leu2160 2165 2170 gaa gct aca cag gat gat tat gta ctt gaa gtg agg cac tttcag 6705 Glu Ala Thr Gln Asp Asp Tyr Val Leu Glu Val Arg His Phe Gln2175 2180 2185 tgt cct aaa tgg cca aat cca gat agc ccc att agt aaa actttt 6750 Cys Pro Lys Trp Pro Asn Pro Asp Ser Pro Ile Ser Lys Thr Phe2190 2195 2200 gaa ctt ata agt gtt ata aaa gaa gaa gct gcc aat agg gatggg 6795 Glu Leu Ile Ser Val Ile Lys Glu Glu Ala Ala Asn Arg Asp Gly2205 2210 2215 cct atg att gtt cat gat gag cat gga gga gtg acg gca ggaact 6840 Pro Met Ile Val His Asp Glu His Gly Gly Val Thr Ala Gly Thr2220 2225 2230 ttc tgt gct ctg aca acc ctt atg cac caa cta gaa aaa gaaaat 6885 Phe Cys Ala Leu Thr Thr Leu Met His Gln Leu Glu Lys Glu Asn2235 2240 2245 tcc gtg gat gtt tac cag gta gcc aag atg atc aat ctg atgagg 6930 Ser Val Asp Val Tyr Gln Val Ala Lys Met Ile Asn Leu Met Arg2250 2255 2260 cca gga gtc ttt gct gac att gag cag tat cag ttt ctc tacaaa 6975 Pro Gly Val Phe Ala Asp Ile Glu Gln Tyr Gln Phe Leu Tyr Lys2265 2270 2275 gtg atc ctc agc ctt gtg agc aca agg cag gaa gag aat ccatcc 7020 Val Ile Leu Ser Leu Val Ser Thr Arg Gln Glu Glu Asn Pro Ser2280 2285 2290 acc tct ctg gac agt aat ggt gca gca ttg cct gat gga aatata 7065 Thr Ser Leu Asp Ser Asn Gly Ala Ala Leu Pro Asp Gly Asn Ile2295 2300 2305 gct gag agc tta gag tct tta gtt taa cacagaaagg ggtgggggga7112 Ala Glu Ser Leu Glu Ser Leu Val 2310 ctcacatctg agcattgttttcctcttcct aaaattaggc aggaaaatca gtctagttct 7172 gttatctgtt gatttcccatcacctgacag taactttcat gacataggat tctgccgcca 7232 aatttatatc attaacaatgtgtgcctttt tgcaagactt gtaatttact tattatgttt 7292 gaactaaaat gattgaattttacagtattt ctaagaatgg aattgtggta tttttttctg 7352 tattgatttt aacagaaaatttcaatttat agaggttagg aattccaaac tacagaaaat 7412 gtttgttttt agtgtcaaatttttagctgt atttgtagca attatcaggt ttgctagaaa 7472 tataactttt aatacagtagcctgtaaata aaacactctt ccatatgata ttcaacattt 7532 tacaactgca gtattcacctaaagtagaaa taatctgtta cttattgtaa atactgccct 7592 agtgtctcca tggaccaaatttatatttat aattgtagat ttttatattt tactactgag 7652 tcaagttttc tagttctgtgtaattgttta gtttaatgac gtagttcatt agctggtctt 7712 actctaccag ttttctgacattgtattgtg ttacctaagt cattaacttt gtttcagcat 7772 gtaattttaa cttttgtggaaaatagaaat accttcattt tgaaagaagt ttttatgaga 7832 ataacacctt accaaacattgttcaaatgg tttttatcca aggaattgca aaaataaata 7892 taaatattgc cattaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaa 7941 6 2314 PRT Homo sapiens gene(1)..(2314) PTP-zeta 6 Met Arg Ile Leu Lys Arg Phe Leu Ala Cys Ile GlnLeu Leu Cys Val 1 5 10 15 Cys Arg Leu Asp Trp Ala Asn Gly Tyr Tyr ArgGln Gln Arg Lys Leu 20 25 30 Val Glu Glu Ile Gly Trp Ser Tyr Thr Gly AlaLeu Asn Gln Lys Asn 35 40 45 Trp Gly Lys Lys Tyr Pro Thr Cys Asn Ser ProLys Gln Ser Pro Ile 50 55 60 Asn Ile Asp Glu Asp Leu Thr Gln Val Asn ValAsn Leu Lys Lys Leu 65 70 75 80 Lys Phe Gln Gly Trp Asp Lys Thr Ser LeuGlu Asn Thr Phe Ile His 85 90 95 Asn Thr Gly Lys Thr Val Glu Ile Asn LeuThr Asn Asp Tyr Arg Val 100 105 110 Ser Gly Gly Val Ser Glu Met Val PheLys Ala Ser Lys Ile Thr Phe 115 120 125 His Trp Gly Lys Cys Asn Met SerSer Asp Gly Ser Glu His Ser Leu 130 135 140 Glu Gly Gln Lys Phe Pro LeuGlu Met Gln Ile Tyr Cys Phe Asp Ala 145 150 155 160 Asp Arg Phe Ser SerPhe Glu Glu Ala Val Lys Gly Lys Gly Lys Leu 165 170 175 Arg Ala Leu SerIle Leu Phe Glu Val Gly Thr Glu Glu Asn Leu Asp 180 185 190 Phe Lys AlaIle Ile Asp Gly Val Glu Ser Val Ser Arg Phe Gly Lys 195 200 205 Gln AlaAla Leu Asp Pro Phe Ile Leu Leu Asn Leu Leu Pro Asn Ser 210 215 220 ThrAsp Lys Tyr Tyr Ile Tyr Asn Gly Ser Leu Thr Ser Pro Pro Cys 225 230 235240 Thr Asp Thr Val Asp Trp Ile Val Phe Lys Asp Thr Val Ser Ile Ser 245250 255 Glu Ser Gln Leu Ala Val Phe Cys Glu Val Leu Thr Met Gln Gln Ser260 265 270 Gly Tyr Val Met Leu Met Asp Tyr Leu Gln Asn Asn Phe Arg GluGln 275 280 285 Gln Tyr Lys Phe Ser Arg Gln Val Phe Ser Ser Tyr Thr GlyLys Glu 290 295 300 Glu Ile His Glu Ala Val Cys Ser Ser Glu Pro Glu AsnVal Gln Ala 305 310 315 320 Asp Pro Glu Asn Tyr Thr Ser Leu Leu Val ThrTrp Glu Arg Pro Arg 325 330 335 Val Val Tyr Asp Thr Met Ile Glu Lys PheAla Val Leu Tyr Gln Gln 340 345 350 Leu Asp Gly Glu Asp Gln Thr Lys HisGlu Phe Leu Thr Asp Gly Tyr 355 360 365 Gln Asp Leu Gly Ala Ile Leu AsnAsn Leu Leu Pro Asn Met Ser Tyr 370 375 380 Val Leu Gln Ile Val Ala IleCys Thr Asn Gly Leu Tyr Gly Lys Tyr 385 390 395 400 Ser Asp Gln Leu IleVal Asp Met Pro Thr Asp Asn Pro Glu Leu Asp 405 410 415 Leu Phe Pro GluLeu Ile Gly Thr Glu Glu Ile Ile Lys Glu Glu Glu 420 425 430 Glu Gly LysAsp Ile Glu Glu Gly Ala Ile Val Asn Pro Gly Arg Asp 435 440 445 Ser AlaThr Asn Gln Ile Arg Lys Lys Glu Pro Gln Ile Ser Thr Thr 450 455 460 ThrHis Tyr Asn Arg Ile Gly Thr Lys Tyr Asn Glu Ala Lys Thr Asn 465 470 475480 Arg Ser Pro Thr Arg Gly Ser Glu Phe Ser Gly Lys Gly Asp Val Pro 485490 495 Asn Thr Ser Leu Asn Ser Thr Ser Gln Pro Val Thr Lys Leu Ala Thr500 505 510 Glu Lys Asp Ile Ser Leu Thr Ser Gln Thr Val Thr Glu Leu ProPro 515 520 525 His Thr Val Glu Gly Thr Ser Ala Ser Leu Asn Asp Gly SerLys Thr 530 535 540 Val Leu Arg Ser Pro His Met Asn Leu Ser Gly Thr AlaGlu Ser Leu 545 550 555 560 Asn Thr Val Ser Ile Thr Glu Tyr Glu Glu GluSer Leu Leu Thr Ser 565 570 575 Phe Lys Leu Asp Thr Gly Ala Glu Asp SerSer Gly Ser Ser Pro Ala 580 585 590 Thr Ser Ala Ile Pro Phe Ile Ser GluAsn Ile Ser Gln Gly Tyr Ile 595 600 605 Phe Ser Ser Glu Asn Pro Glu ThrIle Thr Tyr Asp Val Leu Ile Pro 610 615 620 Glu Ser Ala Arg Asn Ala SerGlu Asp Ser Thr Ser Ser Gly Ser Glu 625 630 635 640 Glu Ser Leu Lys AspPro Ser Met Glu Gly Asn Val Trp Phe Pro Ser 645 650 655 Ser Thr Asp IleThr Ala Gln Pro Asp Val Gly Ser Gly Arg Glu Ser 660 665 670 Phe Leu GlnThr Asn Tyr Thr Glu Ile Arg Val Asp Glu Ser Glu Lys 675 680 685 Thr ThrLys Ser Phe Ser Ala Gly Pro Val Met Ser Gln Gly Pro Ser 690 695 700 ValThr Asp Leu Glu Met Pro His Tyr Ser Thr Phe Ala Tyr Phe Pro 705 710 715720 Thr Glu Val Thr Pro His Ala Phe Thr Pro Ser Ser Arg Gln Gln Asp 725730 735 Leu Val Ser Thr Val Asn Val Val Tyr Ser Gln Thr Thr Gln Pro Val740 745 750 Tyr Asn Gly Glu Thr Pro Leu Gln Pro Ser Tyr Ser Ser Glu ValPhe 755 760 765 Pro Leu Val Thr Pro Leu Leu Leu Asp Asn Gln Ile Leu AsnThr Thr 770 775 780 Pro Ala Ala Ser Ser Ser Asp Ser Ala Leu His Ala ThrPro Val Phe 785 790 795 800 Pro Ser Val Asp Val Ser Phe Glu Ser Ile LeuSer Ser Tyr Asp Gly 805 810 815 Ala Pro Leu Leu Pro Phe Ser Ser Ala SerPhe Ser Ser Glu Leu Phe 820 825 830 Arg His Leu His Thr Val Ser Gln IleLeu Pro Gln Val Thr Ser Ala 835 840 845 Thr Glu Ser Asp Lys Val Pro LeuHis Ala Ser Leu Pro Val Ala Gly 850 855 860 Gly Asp Leu Leu Leu Glu ProSer Leu Ala Gln Tyr Ser Asp Val Leu 865 870 875 880 Ser Thr Thr His AlaAla Ser Glu Thr Leu Glu Phe Gly Ser Glu Ser 885 890 895 Gly Val Leu TyrLys Thr Leu Met Phe Ser Gln Val Glu Pro Pro Ser 900 905 910 Ser Asp AlaMet Met His Ala Arg Ser Ser Gly Pro Glu Pro Ser Tyr 915 920 925 Ala LeuSer Asp Asn Glu Gly Ser Gln His Ile Phe Thr Val Ser Tyr 930 935 940 SerSer Ala Ile Pro Val His Asp Ser Val Gly Val Thr Tyr Gln Gly 945 950 955960 Ser Leu Phe Ser Gly Pro Ser His Ile Pro Ile Pro Lys Ser Ser Leu 965970 975 Ile Thr Pro Thr Ala Ser Leu Leu Gln Pro Thr His Ala Leu Ser Gly980 985 990 Asp Gly Glu Trp Ser Gly Ala Ser Ser Asp Ser Glu Phe Leu LeuPro 995 1000 1005 Asp Thr Asp Gly Leu Thr Ala Leu Asn Ile Ser Ser ProVal Ser 1010 1015 1020 Val Ala Glu Phe Thr Tyr Thr Thr Ser Val Phe GlyAsp Asp Asn 1025 1030 1035 Lys Ala Leu Ser Lys Ser Glu Ile Ile Tyr GlyAsn Glu Thr Glu 1040 1045 1050 Leu Gln Ile Pro Ser Phe Asn Glu Met ValTyr Pro Ser Glu Ser 1055 1060 1065 Thr Val Met Pro Asn Met Tyr Asp AsnVal Asn Lys Leu Asn Ala 1070 1075 1080 Ser Leu Gln Glu Thr Ser Val SerIle Ser Ser Thr Lys Gly Met 1085 1090 1095 Phe Pro Gly Ser Leu Ala HisThr Thr Thr Lys Val Phe Asp His 1100 1105 1110 Glu Ile Ser Gln Val ProGlu Asn Asn Phe Ser Val Gln Pro Thr 1115 1120 1125 His Thr Val Ser GlnAla Ser Gly Asp Thr Ser Leu Lys Pro Val 1130 1135 1140 Leu Ser Ala AsnSer Glu Pro Ala Ser Ser Asp Pro Ala Ser Ser 1145 1150 1155 Glu Met LeuSer Pro Ser Thr Gln Leu Leu Phe Tyr Glu Thr Ser 1160 1165 1170 Ala SerPhe Ser Thr Glu Val Leu Leu Gln Pro Ser Phe Gln Ala 1175 1180 1185 SerAsp Val Asp Thr Leu Leu Lys Thr Val Leu Pro Ala Val Pro 1190 1195 1200Ser Asp Pro Ile Leu Val Glu Thr Pro Lys Val Asp Lys Ile Ser 1205 12101215 Ser Thr Met Leu His Leu Ile Val Ser Asn Ser Ala Ser Ser Glu 12201225 1230 Asn Met Leu His Ser Thr Ser Val Pro Val Phe Asp Val Ser Pro1235 1240 1245 Thr Ser His Met His Ser Ala Ser Leu Gln Gly Leu Thr IleSer 1250 1255 1260 Tyr Ala Ser Glu Lys Tyr Glu Pro Val Leu Leu Lys SerGlu Ser 1265 1270 1275 Ser His Gln Val Val Pro Ser Leu Tyr Ser Asn AspGlu Leu Phe 1280 1285 1290 Gln Thr Ala Asn Leu Glu Ile Asn Gln Ala HisPro Pro Lys Gly 1295 1300 1305 Arg His Val Phe Ala Thr Pro Val Leu SerIle Asp Glu Pro Leu 1310 1315 1320 Asn Thr Leu Ile Asn Lys Leu Ile HisSer Asp Glu Ile Leu Thr 1325 1330 1335 Ser Thr Lys Ser Ser Val Thr GlyLys Val Phe Ala Gly Ile Pro 1340 1345 1350 Thr Val Ala Ser Asp Thr PheVal Ser Thr Asp His Ser Val Pro 1355 1360 1365 Ile Gly Asn Gly His ValAla Ile Thr Ala Val Ser Pro His Arg 1370 1375 1380 Asp Gly Ser Val ThrSer Thr Lys Leu Leu Phe Pro Ser Lys Ala 1385 1390 1395 Thr Ser Glu LeuSer His Ser Ala Lys Ser Asp Ala Gly Leu Val 1400 1405 1410 Gly Gly GlyGlu Asp Gly Asp Thr Asp Asp Asp Gly Asp Asp Asp 1415 1420 1425 Asp AspArg Asp Ser Asp Gly Leu Ser Ile His Lys Cys Met Ser 1430 1435 1440 CysSer Ser Tyr Arg Glu Ser Gln Glu Lys Val Met Asn Asp Ser 1445 1450 1455Asp Thr His Glu Asn Ser Leu Met Asp Gln Asn Asn Pro Ile Ser 1460 14651470 Tyr Ser Leu Ser Glu Asn Ser Glu Glu Asp Asn Arg Val Thr Ser 14751480 1485 Val Ser Ser Asp Ser Gln Thr Gly Met Asp Arg Ser Pro Gly Lys1490 1495 1500 Ser Pro Ser Ala Asn Gly Leu Ser Gln Lys His Asn Asp GlyLys 1505 1510 1515 Glu Glu Asn Asp Ile Gln Thr Gly Ser Ala Leu Leu ProLeu Ser 1520 1525 1530 Pro Glu Ser Lys Ala Trp Ala Val Leu Thr Ser AspGlu Glu Ser 1535 1540 1545 Gly Ser Gly Gln Gly Thr Ser Asp Ser Leu AsnGlu Asn Glu Thr 1550 1555 1560 Ser Thr Asp Phe Ser Phe Ala Asp Thr AsnGlu Lys Asp Ala Asp 1565 1570 1575 Gly Ile Leu Ala Ala Gly Asp Ser GluIle Thr Pro Gly Phe Pro 1580 1585 1590 Gln Ser Pro Thr Ser Ser Val ThrSer Glu Asn Ser Glu Val Phe 1595 1600 1605 His Val Ser Glu Ala Glu AlaSer Asn Ser Ser His Glu Ser Arg 1610 1615 1620 Ile Gly Leu Ala Glu GlyLeu Glu Ser Glu Lys Lys Ala Val Ile 1625 1630 1635 Pro Leu Val Ile ValSer Ala Leu Thr Phe Ile Cys Leu Val Val 1640 1645 1650 Leu Val Gly IleLeu Ile Tyr Trp Arg Lys Cys Phe Gln Thr Ala 1655 1660 1665 His Phe TyrLeu Glu Asp Ser Thr Ser Pro Arg Val Ile Ser Thr 1670 1675 1680 Pro ProThr Pro Ile Phe Pro Ile Ser Asp Asp Val Gly Ala Ile 1685 1690 1695 ProIle Lys His Phe Pro Lys His Val Ala Asp Leu His Ala Ser 1700 1705 1710Ser Gly Phe Thr Glu Glu Phe Glu Thr Leu Lys Glu Phe Tyr Gln 1715 17201725 Glu Val Gln Ser Cys Thr Val Asp Leu Gly Ile Thr Ala Asp Ser 17301735 1740 Ser Asn His Pro Asp Asn Lys His Lys Asn Arg Tyr Ile Asn Ile1745 1750 1755 Val Ala Tyr Asp His Ser Arg Val Lys Leu Ala Gln Leu AlaGlu 1760 1765 1770 Lys Asp Gly Lys Leu Thr Asp Tyr Ile Asn Ala Asn TyrVal Asp 1775 1780 1785 Gly Tyr Asn Arg Pro Lys Ala Tyr Ile Ala Ala GlnGly Pro Leu 1790 1795 1800 Lys Ser Thr Ala Glu Asp Phe Trp Arg Met IleTrp Glu His Asn 1805 1810 1815 Val Glu Val Ile Val Met Ile Thr Asn LeuVal Glu Lys Gly Arg 1820 1825 1830 Arg Lys Cys Asp Gln Tyr Trp Pro AlaAsp Gly Ser Glu Glu Tyr 1835 1840 1845 Gly Asn Phe Leu Val Thr Gln LysSer Val Gln Val Leu Ala Tyr 1850 1855 1860 Tyr Thr Val Arg Asn Phe ThrLeu Arg Asn Thr Lys Ile Lys Lys 1865 1870 1875 Gly Ser Gln Lys Gly ArgPro Ser Gly Arg Val Val Thr Gln Tyr 1880 1885 1890 His Tyr Thr Gln TrpPro Asp Met Gly Val Pro Glu Tyr Ser Leu 1895 1900 1905 Pro Val Leu ThrPhe Val Arg Lys Ala Ala Tyr Ala Lys Arg His 1910 1915 1920 Ala Val GlyPro Val Val Val His Cys Ser Ala Gly Val Gly Arg 1925 1930 1935 Thr GlyThr Tyr Ile Val Leu Asp Ser Met Leu Gln Gln Ile Gln 1940 1945 1950 HisGlu Gly Thr Val Asn Ile Phe Gly Phe Leu Lys His Ile Arg 1955 1960 1965Ser Gln Arg Asn Tyr Leu Val Gln Thr Glu Glu Gln Tyr Val Phe 1970 19751980 Ile His Asp Thr Leu Val Glu Ala Ile Leu Ser Lys Glu Thr Glu 19851990 1995 Val Leu Asp Ser His Ile His Ala Tyr Val Asn Ala Leu Leu Ile2000 2005 2010 Pro Gly Pro Ala Gly Lys Thr Lys Leu Glu Lys Gln Phe GlnLeu 2015 2020 2025 Leu Ser Gln Ser Asn Ile Gln Gln Ser Asp Tyr Ser AlaAla Leu 2030 2035 2040 Lys Gln Cys Asn Arg Glu Lys Asn Arg Thr Ser SerIle Ile Pro 2045 2050 2055 Val Glu Arg Ser Arg Val Gly Ile Ser Ser LeuSer Gly Glu Gly 2060 2065 2070 Thr Asp Tyr Ile Asn Ala Ser Tyr Ile MetGly Tyr Tyr Gln Ser 2075 2080 2085 Asn Glu Phe Ile Ile Thr Gln His ProLeu Leu His Thr Ile Lys 2090 2095 2100 Asp Phe Trp Arg Met Ile Trp AspHis Asn Ala Gln Leu Val Val 2105 2110 2115 Met Ile Pro Asp Gly Gln AsnMet Ala Glu Asp Glu Phe Val Tyr 2120 2125 2130 Trp Pro Asn Lys Asp GluPro Ile Asn Cys Glu Ser Phe Lys Val 2135 2140 2145 Thr Leu Met Ala GluGlu His Lys Cys Leu Ser Asn Glu Glu Lys 2150 2155 2160 Leu Ile Ile GlnAsp Phe Ile Leu Glu Ala Thr Gln Asp Asp Tyr 2165 2170 2175 Val Leu GluVal Arg His Phe Gln Cys Pro Lys Trp Pro Asn Pro 2180 2185 2190 Asp SerPro Ile Ser Lys Thr Phe Glu Leu Ile Ser Val Ile Lys 2195 2200 2205 GluGlu Ala Ala Asn Arg Asp Gly Pro Met Ile Val His Asp Glu 2210 2215 2220His Gly Gly Val Thr Ala Gly Thr Phe Cys Ala Leu Thr Thr Leu 2225 22302235 Met His Gln Leu Glu Lys Glu Asn Ser Val Asp Val Tyr Gln Val 22402245 2250 Ala Lys Met Ile Asn Leu Met Arg Pro Gly Val Phe Ala Asp Ile2255 2260 2265 Glu Gln Tyr Gln Phe Leu Tyr Lys Val Ile Leu Ser Leu ValSer 2270 2275 2280 Thr Arg Gln Glu Glu Asn Pro Ser Thr Ser Leu Asp SerAsn Gly 2285 2290 2295 Ala Ala Leu Pro Asp Gly Asn Ile Ala Glu Ser LeuGlu Ser Leu 2300 2305 2310 Val 7 1518 DNA Homo sapiens gene (1)..(1518)Angiopoietin-like 2 (ANGPTL2), mRNA 7 aaccaccatt ttgcaaggac c atg aggcca ctg tgc gtg aca tgc tgg tgg 51 Met Arg Pro Leu Cys Val Thr Cys TrpTrp 1 5 10 ctc gga ctg ctg gct gcc atg gga gct gtt gca ggc cag gag gacggt 99 Leu Gly Leu Leu Ala Ala Met Gly Ala Val Ala Gly Gln Glu Asp Gly15 20 25 ttt gag ggc act gag gag ggc tcg cca aga gag ttc att tac cta aac147 Phe Glu Gly Thr Glu Glu Gly Ser Pro Arg Glu Phe Ile Tyr Leu Asn 3035 40 agg tac aag cgg gcg ggc gag tcc cag gac aag tgc acc tac acc ttc195 Arg Tyr Lys Arg Ala Gly Glu Ser Gln Asp Lys Cys Thr Tyr Thr Phe 4550 55 att gtg ccc cag cag cgg gtc acg ggt gcc atc tgc gtc aac tcc aag243 Ile Val Pro Gln Gln Arg Val Thr Gly Ala Ile Cys Val Asn Ser Lys 6065 70 gag cct gag gtg ctt ctg gag aac cga gtg cat aag cag gag cta gag291 Glu Pro Glu Val Leu Leu Glu Asn Arg Val His Lys Gln Glu Leu Glu 7580 85 90 ctg ctc aac aat gag ctg ctc aag cag aag cgg cag atc gag aca ctg339 Leu Leu Asn Asn Glu Leu Leu Lys Gln Lys Arg Gln Ile Glu Thr Leu 95100 105 cag cag ctg gtg gag gtg gac ggc ggc att gtg agc gag gtg aag ctg387 Gln Gln Leu Val Glu Val Asp Gly Gly Ile Val Ser Glu Val Lys Leu 110115 120 ctg cgc aag gag agc cgc aac atg aac tcg cgg gtc acg cag ctc tac435 Leu Arg Lys Glu Ser Arg Asn Met Asn Ser Arg Val Thr Gln Leu Tyr 125130 135 atg cag ctc ctg cac gag atc atc cgc aag cgg gac aac gcg ttg gag483 Met Gln Leu Leu His Glu Ile Ile Arg Lys Arg Asp Asn Ala Leu Glu 140145 150 ctc tcc cag ctg gag aac agg atc ctg aac cag aca gcc gac atg ctg531 Leu Ser Gln Leu Glu Asn Arg Ile Leu Asn Gln Thr Ala Asp Met Leu 155160 165 170 cag ctg gcc agc aag tac aag gac ctg gag cac aag tac cag cacctg 579 Gln Leu Ala Ser Lys Tyr Lys Asp Leu Glu His Lys Tyr Gln His Leu175 180 185 gcc aca ctg gcc cac aac caa tca gag atc atc gcg cag ctt gaggag 627 Ala Thr Leu Ala His Asn Gln Ser Glu Ile Ile Ala Gln Leu Glu Glu190 195 200 cac tgc cag agg gtg ccc tcg gcc agg ccc gtc ccc cag cca cccccc 675 His Cys Gln Arg Val Pro Ser Ala Arg Pro Val Pro Gln Pro Pro Pro205 210 215 gct gcc ccg ccc cgg gtc tac caa cca ccc acc tac aac cgc atcatc 723 Ala Ala Pro Pro Arg Val Tyr Gln Pro Pro Thr Tyr Asn Arg Ile Ile220 225 230 aac cag atc tct acc aac gag atc cag agt gac cag aac ctg aaggtg 771 Asn Gln Ile Ser Thr Asn Glu Ile Gln Ser Asp Gln Asn Leu Lys Val235 240 245 250 ctg cca ccc cct ctg ccc act atg ccc act ctc acc agc ctccca tct 819 Leu Pro Pro Pro Leu Pro Thr Met Pro Thr Leu Thr Ser Leu ProSer 255 260 265 tcc acc gac aag ccg tcg ggc cca tgg aga gac tgc ctg caggcc ctg 867 Ser Thr Asp Lys Pro Ser Gly Pro Trp Arg Asp Cys Leu Gln AlaLeu 270 275 280 gag gat ggc cac gac acc agc tcc atc tac ctg gtg aag ccggag aac 915 Glu Asp Gly His Asp Thr Ser Ser Ile Tyr Leu Val Lys Pro GluAsn 285 290 295 acc aac cgc ctc atg cag gtg tgg tgc gac cag aga cac gacccc ggg 963 Thr Asn Arg Leu Met Gln Val Trp Cys Asp Gln Arg His Asp ProGly 300 305 310 ggc tgg acc gtc atc cag aga cgc ctg gat ggc tct gtt aacttc ttc 1011 Gly Trp Thr Val Ile Gln Arg Arg Leu Asp Gly Ser Val Asn PhePhe 315 320 325 330 agg aac tgg gag acg tac aag caa ggg ttt ggg aac attgat ggc gaa 1059 Arg Asn Trp Glu Thr Tyr Lys Gln Gly Phe Gly Asn Ile AspGly Glu 335 340 345 tac tgg ctg ggc ctg gag aac att tac tgg ctg acg aaccaa ggc aac 1107 Tyr Trp Leu Gly Leu Glu Asn Ile Tyr Trp Leu Thr Asn GlnGly Asn 350 355 360 tac aaa ctc ctg gtg acc atg gag gac tgg tcc ggc cgcaaa gtc ttt 1155 Tyr Lys Leu Leu Val Thr Met Glu Asp Trp Ser Gly Arg LysVal Phe 365 370 375 gca gaa tac gcc agt ttc cgc ctg gaa cct gag agc gagtat tat aag 1203 Ala Glu Tyr Ala Ser Phe Arg Leu Glu Pro Glu Ser Glu TyrTyr Lys 380 385 390 ctg cgg ctg ggg cgc tac cat ggc aat gcg ggt gac tccttt aca tgg 1251 Leu Arg Leu Gly Arg Tyr His Gly Asn Ala Gly Asp Ser PheThr Trp 395 400 405 410 cac aac ggc aag cag ttc acc acc ctg gac aga gatcat gat gtc tac 1299 His Asn Gly Lys Gln Phe Thr Thr Leu Asp Arg Asp HisAsp Val Tyr 415 420 425 aca gga aac tgt gcc cac tac cag aag gga ggc tggtgg tat aac gcc 1347 Thr Gly Asn Cys Ala His Tyr Gln Lys Gly Gly Trp TrpTyr Asn Ala 430 435 440 tgt gcc cac tcc aac ctc aac ggg gtc tgg tac cgcggg ggc cat tac 1395 Cys Ala His Ser Asn Leu Asn Gly Val Trp Tyr Arg GlyGly His Tyr 445 450 455 cgg agc cgc tac cag gac gga gtc tac tgg gct gagttc cga gga ggc 1443 Arg Ser Arg Tyr Gln Asp Gly Val Tyr Trp Ala Glu PheArg Gly Gly 460 465 470 tct tac tca ctc aag aaa gtg gtg atg atg atc cgaccg aac ccc aac 1491 Ser Tyr Ser Leu Lys Lys Val Val Met Met Ile Arg ProAsn Pro Asn 475 480 485 490 acc ttc cac taa gccagctccc cctcc 1518 ThrPhe His 8 493 PRT Homo sapiens gene (1)..(493) Angiopoietin-like 2(ANGPTL2), protein 8 Met Arg Pro Leu Cys Val Thr Cys Trp Trp Leu Gly LeuLeu Ala Ala 1 5 10 15 Met Gly Ala Val Ala Gly Gln Glu Asp Gly Phe GluGly Thr Glu Glu 20 25 30 Gly Ser Pro Arg Glu Phe Ile Tyr Leu Asn Arg TyrLys Arg Ala Gly 35 40 45 Glu Ser Gln Asp Lys Cys Thr Tyr Thr Phe Ile ValPro Gln Gln Arg 50 55 60 Val Thr Gly Ala Ile Cys Val Asn Ser Lys Glu ProGlu Val Leu Leu 65 70 75 80 Glu Asn Arg Val His Lys Gln Glu Leu Glu LeuLeu Asn Asn Glu Leu 85 90 95 Leu Lys Gln Lys Arg Gln Ile Glu Thr Leu GlnGln Leu Val Glu Val 100 105 110 Asp Gly Gly Ile Val Ser Glu Val Lys LeuLeu Arg Lys Glu Ser Arg 115 120 125 Asn Met Asn Ser Arg Val Thr Gln LeuTyr Met Gln Leu Leu His Glu 130 135 140 Ile Ile Arg Lys Arg Asp Asn AlaLeu Glu Leu Ser Gln Leu Glu Asn 145 150 155 160 Arg Ile Leu Asn Gln ThrAla Asp Met Leu Gln Leu Ala Ser Lys Tyr 165 170 175 Lys Asp Leu Glu HisLys Tyr Gln His Leu Ala Thr Leu Ala His Asn 180 185 190 Gln Ser Glu IleIle Ala Gln Leu Glu Glu His Cys Gln Arg Val Pro 195 200 205 Ser Ala ArgPro Val Pro Gln Pro Pro Pro Ala Ala Pro Pro Arg Val 210 215 220 Tyr GlnPro Pro Thr Tyr Asn Arg Ile Ile Asn Gln Ile Ser Thr Asn 225 230 235 240Glu Ile Gln Ser Asp Gln Asn Leu Lys Val Leu Pro Pro Pro Leu Pro 245 250255 Thr Met Pro Thr Leu Thr Ser Leu Pro Ser Ser Thr Asp Lys Pro Ser 260265 270 Gly Pro Trp Arg Asp Cys Leu Gln Ala Leu Glu Asp Gly His Asp Thr275 280 285 Ser Ser Ile Tyr Leu Val Lys Pro Glu Asn Thr Asn Arg Leu MetGln 290 295 300 Val Trp Cys Asp Gln Arg His Asp Pro Gly Gly Trp Thr ValIle Gln 305 310 315 320 Arg Arg Leu Asp Gly Ser Val Asn Phe Phe Arg AsnTrp Glu Thr Tyr 325 330 335 Lys Gln Gly Phe Gly Asn Ile Asp Gly Glu TyrTrp Leu Gly Leu Glu 340 345 350 Asn Ile Tyr Trp Leu Thr Asn Gln Gly AsnTyr Lys Leu Leu Val Thr 355 360 365 Met Glu Asp Trp Ser Gly Arg Lys ValPhe Ala Glu Tyr Ala Ser Phe 370 375 380 Arg Leu Glu Pro Glu Ser Glu TyrTyr Lys Leu Arg Leu Gly Arg Tyr 385 390 395 400 His Gly Asn Ala Gly AspSer Phe Thr Trp His Asn Gly Lys Gln Phe 405 410 415 Thr Thr Leu Asp ArgAsp His Asp Val Tyr Thr Gly Asn Cys Ala His 420 425 430 Tyr Gln Lys GlyGly Trp Trp Tyr Asn Ala Cys Ala His Ser Asn Leu 435 440 445 Asn Gly ValTrp Tyr Arg Gly Gly His Tyr Arg Ser Arg Tyr Gln Asp 450 455 460 Gly ValTyr Trp Ala Glu Phe Arg Gly Gly Ser Tyr Ser Leu Lys Lys 465 470 475 480Val Val Met Met Ile Arg Pro Asn Pro Asn Thr Phe His 485 490 9 2133 DNAHomo sapiens gene (1)..(2133) Human SPARC/osteonectin mRNA 9 cgggagagcgcgctctgcct gccgcctgcc tgcctgccac tgagggttcc cagcacc 57 atg agg gcc tggatc ttc ttt ctc ctt tgc ctg gcc ggg agg gcc ttg 105 Met Arg Ala Trp IlePhe Phe Leu Leu Cys Leu Ala Gly Arg Ala Leu 1 5 10 15 gca gcc cct cagcaa gaa gcc ctg cct gat gag aca gag gtg gtg gaa 153 Ala Ala Pro Gln GlnGlu Ala Leu Pro Asp Glu Thr Glu Val Val Glu 20 25 30 gaa act gtg gca gaggtg act gag gta tct gtg gga gct aat cct gtc 201 Glu Thr Val Ala Glu ValThr Glu Val Ser Val Gly Ala Asn Pro Val 35 40 45 cag gtg gaa gta gga gaattt gat gat ggt gca gag gaa acc gaa gag 249 Gln Val Glu Val Gly Glu PheAsp Asp Gly Ala Glu Glu Thr Glu Glu 50 55 60 gag gtg gtg gcg gaa aat ccctgc cag aac cac cac tgc aaa cac ggc 297 Glu Val Val Ala Glu Asn Pro CysGln Asn His His Cys Lys His Gly 65 70 75 80 aag gtg tgc gag ctg gat gagaac aac acc ccc atg tgc gtg tgc cag 345 Lys Val Cys Glu Leu Asp Glu AsnAsn Thr Pro Met Cys Val Cys Gln 85 90 95 gac ccc acc agc tgc cca gcc cccatt ggc gag ttt gag aag gtg tgc 393 Asp Pro Thr Ser Cys Pro Ala Pro IleGly Glu Phe Glu Lys Val Cys 100 105 110 agc aat gac aac aag acc ttc gactct tcc tgc cac ttc ttt gcc aca 441 Ser Asn Asp Asn Lys Thr Phe Asp SerSer Cys His Phe Phe Ala Thr 115 120 125 aag tgc acc ctg gag ggc acc aagaag ggc cac aag ctc cac ctg gac 489 Lys Cys Thr Leu Glu Gly Thr Lys LysGly His Lys Leu His Leu Asp 130 135 140 tac atc ggg cct tgc aaa tac atcccc cct tgc ctg gac tct gag ctg 537 Tyr Ile Gly Pro Cys Lys Tyr Ile ProPro Cys Leu Asp Ser Glu Leu 145 150 155 160 acc gaa ttc ccc ctg cgc atgcgg gac tgg ctc aag aac gtc ctg gtc 585 Thr Glu Phe Pro Leu Arg Met ArgAsp Trp Leu Lys Asn Val Leu Val 165 170 175 acc ctg tat gag agg gat gaggac aac aac ctt ctg act gag aag cag 633 Thr Leu Tyr Glu Arg Asp Glu AspAsn Asn Leu Leu Thr Glu Lys Gln 180 185 190 aag ctg cgg gtg aag aag atccat gag aat gag aag cgc ctg gag gca 681 Lys Leu Arg Val Lys Lys Ile HisGlu Asn Glu Lys Arg Leu Glu Ala 195 200 205 gga gac cac ccc gtg gag ctgctg gcc cgg gac ttc gag aag aac tat 729 Gly Asp His Pro Val Glu Leu LeuAla Arg Asp Phe Glu Lys Asn Tyr 210 215 220 aac atg tac atc ttc cct gtacac tgg cag ttc ggc cag ctg gac cag 777 Asn Met Tyr Ile Phe Pro Val HisTrp Gln Phe Gly Gln Leu Asp Gln 225 230 235 240 cac ccc att gac ggg tacctc tcc cac acc gag ctg gct cca ctg cgt 825 His Pro Ile Asp Gly Tyr LeuSer His Thr Glu Leu Ala Pro Leu Arg 245 250 255 gct ccc ctc atc ccc atggag cat tgc acc acc cgc ttt ttc gag acc 873 Ala Pro Leu Ile Pro Met GluHis Cys Thr Thr Arg Phe Phe Glu Thr 260 265 270 tgt gac ctg gac aat gacaag tac atc gcc ctg gat gag tgg gcc ggc 921 Cys Asp Leu Asp Asn Asp LysTyr Ile Ala Leu Asp Glu Trp Ala Gly 275 280 285 tgc ttc ggc atc aag cagaag gat atc gac aag gat ctt gtg atc taa 969 Cys Phe Gly Ile Lys Gln LysAsp Ile Asp Lys Asp Leu Val Ile 290 295 300 atccactcct tccacagtaccggattctct ctttaaccct ccccttcgtg tttcccccaa 1029 tgtttaaaat gtttggatggtttgttgttc tgcctggaga caaggtgcta acatagattt 1089 aagtgaatac attaacggtgctaaaaatga aaattctaac ccaagacatg acattcttag 1149 ctgtaactta actattaaggccttttccac acgcattaat agtcccattt ttctcttgcc 1209 atttgtagct ttgcccattgtcttattggc acatgggtgg acacggatct gctgggctct 1269 gccttaaaca cacattgcagcttcaacttt tctctttagt gttctgtttg aaactaatac 1329 ttaccgagtc agactttgtgttcatttcat ttcagggtct tggctgcctg tgggcttccc 1389 caggtggcct ggaggtgggcaaagggaagt aacagacaca cgatgttgtc aaggatggtt 1449 ttgggactag aggctcagtggtgggagaga tccctgcaga atccaccaac cagaacgtgg 1509 tttgcctgag gctgtaactgagagaaagat tctggggctg tcttatgaaa atatagacat 1569 tctcacataa gcccagttcatcaccatttc ctcctttacc tttcagtgca gtttcttttc 1629 acattaggct gttggttcaaacttttggga gcacggactg tcagttctct gggaagtggt 1689 cagcgcatcc tgcagggcttctcctcctct gtcttttgga gaaccagggc tcttctcagg 1749 ggctctaggg actgccaggctgtttcagcc aggaaggcca aaatcaagag tgagatgtag 1809 aaagttgtaa aatagaaaaagtggagttgg tgaatcggtt gttctttcct cacatttgga 1869 tgattgtcat aaggtttttagcatgttcct ccttttcttc accctcccct ttgttcttct 1929 attaatcaag agaaacttcaaagttaatgg gatggtcgga tctcacaggc tgagaactcg 1989 ttcacctcca agcatttcatgaaaaagctg cttcttatta atcatacaaa ctctcaccat 2049 gatgtgaaga gtttcacaaatctttcaaaa taaaaagtaa tgacttagaa actgaaaaaa 2109 aaaaaaaaaa aaaaaaaaaaaaaa 2133 10 303 PRT Homo sapiens SIGNAL (1)..(17) 10 Met Arg Ala TrpIle Phe Phe Leu Leu Cys Leu Ala Gly Arg Ala Leu 1 5 10 15 Ala Ala ProGln Gln Glu Ala Leu Pro Asp Glu Thr Glu Val Val Glu 20 25 30 Glu Thr ValAla Glu Val Thr Glu Val Ser Val Gly Ala Asn Pro Val 35 40 45 Gln Val GluVal Gly Glu Phe Asp Asp Gly Ala Glu Glu Thr Glu Glu 50 55 60 Glu Val ValAla Glu Asn Pro Cys Gln Asn His His Cys Lys His Gly 65 70 75 80 Lys ValCys Glu Leu Asp Glu Asn Asn Thr Pro Met Cys Val Cys Gln 85 90 95 Asp ProThr Ser Cys Pro Ala Pro Ile Gly Glu Phe Glu Lys Val Cys 100 105 110 SerAsn Asp Asn Lys Thr Phe Asp Ser Ser Cys His Phe Phe Ala Thr 115 120 125Lys Cys Thr Leu Glu Gly Thr Lys Lys Gly His Lys Leu His Leu Asp 130 135140 Tyr Ile Gly Pro Cys Lys Tyr Ile Pro Pro Cys Leu Asp Ser Glu Leu 145150 155 160 Thr Glu Phe Pro Leu Arg Met Arg Asp Trp Leu Lys Asn Val LeuVal 165 170 175 Thr Leu Tyr Glu Arg Asp Glu Asp Asn Asn Leu Leu Thr GluLys Gln 180 185 190 Lys Leu Arg Val Lys Lys Ile His Glu Asn Glu Lys ArgLeu Glu Ala 195 200 205 Gly Asp His Pro Val Glu Leu Leu Ala Arg Asp PheGlu Lys Asn Tyr 210 215 220 Asn Met Tyr Ile Phe Pro Val His Trp Gln PheGly Gln Leu Asp Gln 225 230 235 240 His Pro Ile Asp Gly Tyr Leu Ser HisThr Glu Leu Ala Pro Leu Arg 245 250 255 Ala Pro Leu Ile Pro Met Glu HisCys Thr Thr Arg Phe Phe Glu Thr 260 265 270 Cys Asp Leu Asp Asn Asp LysTyr Ile Ala Leu Asp Glu Trp Ala Gly 275 280 285 Cys Phe Gly Ile Lys GlnLys Asp Ile Asp Lys Asp Leu Val Ile 290 295 300 11 4586 DNA Homo sapiensgene (1)..(4586) mRNA for met proto-oncogene 11 gaattccgcc ctcgccgcccgcggcgcccc gagcgctttg tgagcagatg cggagccgag 60 tggagggcgc gagccagatgcggggcgaca gctgacttgc tgagaggagg cggggaggcg 120 cggagcgcgc gtgtggtccttgcgccgctg acttctccac tggttcctgg gcaccgaaag 180 ataaacctct cata atg aaggcc ccc gct gtg ctt gca cct ggc atc ctc 230 Met Lys Ala Pro Ala Val LeuAla Pro Gly Ile Leu 1 5 10 gtg ctc ctg ttt acc ttg gtg cag agg agc aatggg gag tgt aaa gag 278 Val Leu Leu Phe Thr Leu Val Gln Arg Ser Asn GlyGlu Cys Lys Glu 15 20 25 gca cta gca aag tcc gag atg aat gtg aat atg aagtat cag ctt ccc 326 Ala Leu Ala Lys Ser Glu Met Asn Val Asn Met Lys TyrGln Leu Pro 30 35 40 aac ttc acc gcg gaa aca ccc atc cag aat gtc att ctacat gag cat 374 Asn Phe Thr Ala Glu Thr Pro Ile Gln Asn Val Ile Leu HisGlu His 45 50 55 60 cac att ttc ctt ggt gcc act aac tac att tat gtt ttaaat gag gaa 422 His Ile Phe Leu Gly Ala Thr Asn Tyr Ile Tyr Val Leu AsnGlu Glu 65 70 75 gac ctt cag aag gtt gct gag tac aag act ggg cct gtg ctggaa cac 470 Asp Leu Gln Lys Val Ala Glu Tyr Lys Thr Gly Pro Val Leu GluHis 80 85 90 cca gat tgt ttc cca tgt cag gac tgc agc agc aaa gcc aat ttatca 518 Pro Asp Cys Phe Pro Cys Gln Asp Cys Ser Ser Lys Ala Asn Leu Ser95 100 105 gga ggt gtt tgg aaa gat aac atc aac atg gct cta gtt gtc gacacc 566 Gly Gly Val Trp Lys Asp Asn Ile Asn Met Ala Leu Val Val Asp Thr110 115 120 tac tat gat gat caa ctc att agc tgt ggc agc gtc aac aga gggacc 614 Tyr Tyr Asp Asp Gln Leu Ile Ser Cys Gly Ser Val Asn Arg Gly Thr125 130 135 140 tgc cag cga cat gtc ttt ccc cac aat cat act gct gac atacag tcg 662 Cys Gln Arg His Val Phe Pro His Asn His Thr Ala Asp Ile GlnSer 145 150 155 gag gtt cac tgc ata ttc tcc cca cag ata gaa gag ccc agccag tgt 710 Glu Val His Cys Ile Phe Ser Pro Gln Ile Glu Glu Pro Ser GlnCys 160 165 170 cct gac tgt gtg gtg agc gcc ctg gga gcc aaa gtc ctt tcatct gta 758 Pro Asp Cys Val Val Ser Ala Leu Gly Ala Lys Val Leu Ser SerVal 175 180 185 aag gac cgg ttc atc aac ttc ttt gta ggc aat acc ata aattct tct 806 Lys Asp Arg Phe Ile Asn Phe Phe Val Gly Asn Thr Ile Asn SerSer 190 195 200 tat ttc cca gat cat cca ttg cat tcg ata tca gtg aga aggcta aag 854 Tyr Phe Pro Asp His Pro Leu His Ser Ile Ser Val Arg Arg LeuLys 205 210 215 220 gaa acg aaa gat ggt ttt atg ttt ttg acg gac cag tcctac att gat 902 Glu Thr Lys Asp Gly Phe Met Phe Leu Thr Asp Gln Ser TyrIle Asp 225 230 235 gtt tta cct gag ttc aga gat tct tac ccc att aag tatgtc cat gcc 950 Val Leu Pro Glu Phe Arg Asp Ser Tyr Pro Ile Lys Tyr ValHis Ala 240 245 250 ttt gaa agc aac aat ttt att tac ttc ttg acg gtc caaagg gaa act 998 Phe Glu Ser Asn Asn Phe Ile Tyr Phe Leu Thr Val Gln ArgGlu Thr 255 260 265 cta gat gct cag act ttt cac aca aga ata atc agg ttctgt tcc ata 1046 Leu Asp Ala Gln Thr Phe His Thr Arg Ile Ile Arg Phe CysSer Ile 270 275 280 aac tct gga ttg cat tcc tac atg gaa atg cct ctg gagtgt att ctc 1094 Asn Ser Gly Leu His Ser Tyr Met Glu Met Pro Leu Glu CysIle Leu 285 290 295 300 aca gaa aag aga aaa aag aga tcc aca aag aag gaagtg ttt aat ata 1142 Thr Glu Lys Arg Lys Lys Arg Ser Thr Lys Lys Glu ValPhe Asn Ile 305 310 315 ctt cag gct gcg tat gtc agc aag cct ggg gcc cagctt gct aga caa 1190 Leu Gln Ala Ala Tyr Val Ser Lys Pro Gly Ala Gln LeuAla Arg Gln 320 325 330 ata gga gcc agc ctg aat gat gac att ctt ttc ggggtg ttc gca caa 1238 Ile Gly Ala Ser Leu Asn Asp Asp Ile Leu Phe Gly ValPhe Ala Gln 335 340 345 agc aag cca gat tct gcc gaa cca atg gat cga tctgcc atg tgt gca 1286 Ser Lys Pro Asp Ser Ala Glu Pro Met Asp Arg Ser AlaMet Cys Ala 350 355 360 ttc cct atc aaa tat gtc aac gac ttc ttc aac aagatc gtc aac aaa 1334 Phe Pro Ile Lys Tyr Val Asn Asp Phe Phe Asn Lys IleVal Asn Lys 365 370 375 380 aac aat gtg aga tgt ctc cag cat ttt tac ggaccc aat cat gag cac 1382 Asn Asn Val Arg Cys Leu Gln His Phe Tyr Gly ProAsn His Glu His 385 390 395 tgc ttt aat agg aca ctt ctg aga aat tca tcaggc tgt gaa gcg cgc 1430 Cys Phe Asn Arg Thr Leu Leu Arg Asn Ser Ser GlyCys Glu Ala Arg 400 405 410 cgt gat gaa tat cga aca gag ttt acc aca gctttg cag cgc gtt gac 1478 Arg Asp Glu Tyr Arg Thr Glu Phe Thr Thr Ala LeuGln Arg Val Asp 415 420 425 tta ttc atg ggt caa ttc agc gaa gtc ctc ttaaca tct ata tcc acc 1526 Leu Phe Met Gly Gln Phe Ser Glu Val Leu Leu ThrSer Ile Ser Thr 430 435 440 ttc att aaa gga gac ctc acc ata gct aat cttggg aca tca gag ggt 1574 Phe Ile Lys Gly Asp Leu Thr Ile Ala Asn Leu GlyThr Ser Glu Gly 445 450 455 460 cgc ttc atg cag gtt gtg gtt tct cga tcagga cca tca acc cct cat 1622 Arg Phe Met Gln Val Val Val Ser Arg Ser GlyPro Ser Thr Pro His 465 470 475 gtg aat ttt ctc ctg gac tcc cat cca gtgtct cca gaa gtg att gtg 1670 Val Asn Phe Leu Leu Asp Ser His Pro Val SerPro Glu Val Ile Val 480 485 490 gag cat aca tta aac caa aat ggc tac acactg gtt atc act ggg aag 1718 Glu His Thr Leu Asn Gln Asn Gly Tyr Thr LeuVal Ile Thr Gly Lys 495 500 505 aag atc acg aag atc cca ttg aat ggc ttgggc tgc aga cat ttc cag 1766 Lys Ile Thr Lys Ile Pro Leu Asn Gly Leu GlyCys Arg His Phe Gln 510 515 520 tcc tgc agt caa tgc ctc tct gcc cca cccttt gtt cag tgt ggc tgg 1814 Ser Cys Ser Gln Cys Leu Ser Ala Pro Pro PheVal Gln Cys Gly Trp 525 530 535 540 tgc cac gac aaa tgt gtg cga tcg gaggaa tgc ctg agc ggg aca tgg 1862 Cys His Asp Lys Cys Val Arg Ser Glu GluCys Leu Ser Gly Thr Trp 545 550 555 act caa cag atc tgt ctg cct gca atctac aag gtt ttc cca aat agt 1910 Thr Gln Gln Ile Cys Leu Pro Ala Ile TyrLys Val Phe Pro Asn Ser 560 565 570 gca ccc ctt gaa gga ggg aca agg ctgacc ata tgt ggc tgg gac ttt 1958 Ala Pro Leu Glu Gly Gly Thr Arg Leu ThrIle Cys Gly Trp Asp Phe 575 580 585 gga ttt cgg agg aat aat aaa ttt gattta aag aaa act aga gtt ctc 2006 Gly Phe Arg Arg Asn Asn Lys Phe Asp LeuLys Lys Thr Arg Val Leu 590 595 600 ctt gga aat gag agc tgc acc ttg acttta agt gag agc acg atg aat 2054 Leu Gly Asn Glu Ser Cys Thr Leu Thr LeuSer Glu Ser Thr Met Asn 605 610 615 620 aca ttg aaa tgc aca gtt ggt cctgcc atg aat aag cat ttc aat atg 2102 Thr Leu Lys Cys Thr Val Gly Pro AlaMet Asn Lys His Phe Asn Met 625 630 635 tcc ata att att tca aat ggc cacggg aca aca caa tac agt aca ttc 2150 Ser Ile Ile Ile Ser Asn Gly His GlyThr Thr Gln Tyr Ser Thr Phe 640 645 650 tcc tat gtg gat cct gta ata acaagt att tcg ccg aaa tac ggt cct 2198 Ser Tyr Val Asp Pro Val Ile Thr SerIle Ser Pro Lys Tyr Gly Pro 655 660 665 atg gct ggt ggc act tta ctt acttta act gga aat tac cta aac agt 2246 Met Ala Gly Gly Thr Leu Leu Thr LeuThr Gly Asn Tyr Leu Asn Ser 670 675 680 ggg aat tct aga cac att tca attggt gga aaa aca tgt act tta aaa 2294 Gly Asn Ser Arg His Ile Ser Ile GlyGly Lys Thr Cys Thr Leu Lys 685 690 695 700 agt gtg tca aac agt att cttgaa tgt tat acc cca gcc caa acc att 2342 Ser Val Ser Asn Ser Ile Leu GluCys Tyr Thr Pro Ala Gln Thr Ile 705 710 715 tca act gag ttt gct gtt aaattg aaa att gac tta gcc aac cga gag 2390 Ser Thr Glu Phe Ala Val Lys LeuLys Ile Asp Leu Ala Asn Arg Glu 720 725 730 aca agc atc ttc agt tac cgtgaa gat ccc att gtc tat gaa att cat 2438 Thr Ser Ile Phe Ser Tyr Arg GluAsp Pro Ile Val Tyr Glu Ile His 735 740 745 cca acc aaa tct ttt att agtggt ggg agc aca ata aca ggt gtt ggg 2486 Pro Thr Lys Ser Phe Ile Ser GlyGly Ser Thr Ile Thr Gly Val Gly 750 755 760 aaa aac ctg aat tca gtt agtgtc ccg aga atg gtc ata aat gtg cat 2534 Lys Asn Leu Asn Ser Val Ser ValPro Arg Met Val Ile Asn Val His 765 770 775 780 gaa gca gga agg aac tttaca gtg gca tgt caa cat cgc tct aat tca 2582 Glu Ala Gly Arg Asn Phe ThrVal Ala Cys Gln His Arg Ser Asn Ser 785 790 795 gag ata atc tgt tgt accact cct tcc ctg caa cag ctg aat ctg caa 2630 Glu Ile Ile Cys Cys Thr ThrPro Ser Leu Gln Gln Leu Asn Leu Gln 800 805 810 ctc ccc ctg aaa acc aaagcc ttt ttc atg tta gat ggg atc ctt tcc 2678 Leu Pro Leu Lys Thr Lys AlaPhe Phe Met Leu Asp Gly Ile Leu Ser 815 820 825 aaa tac ttt gat ctc atttat gta cat aat cct gtg ttt aag cct ttt 2726 Lys Tyr Phe Asp Leu Ile TyrVal His Asn Pro Val Phe Lys Pro Phe 830 835 840 gaa aag cca gtg atg atctca atg ggc aat gaa aat gta ctg gaa att 2774 Glu Lys Pro Val Met Ile SerMet Gly Asn Glu Asn Val Leu Glu Ile 845 850 855 860 aag gga aat gat attgac cct gaa gca gtt aaa ggt gaa gtg tta aaa 2822 Lys Gly Asn Asp Ile AspPro Glu Ala Val Lys Gly Glu Val Leu Lys 865 870 875 gtt gga aat aag agctgt gag aat ata cac tta cat tct gaa gcc gtt 2870 Val Gly Asn Lys Ser CysGlu Asn Ile His Leu His Ser Glu Ala Val 880 885 890 tta tgc acg gtc cccaat gac ctg ctg aaa ttg aac agc gag cta aat 2918 Leu Cys Thr Val Pro AsnAsp Leu Leu Lys Leu Asn Ser Glu Leu Asn 895 900 905 ata gag tgg aag caagca att tct tca acc gtc ctt gga aaa gta ata 2966 Ile Glu Trp Lys Gln AlaIle Ser Ser Thr Val Leu Gly Lys Val Ile 910 915 920 gtt caa cca gat cagaat ttc aca gga ttg att gct ggt gtt gtc tca 3014 Val Gln Pro Asp Gln AsnPhe Thr Gly Leu Ile Ala Gly Val Val Ser 925 930 935 940 ata tca aca gcactg tta tta cta ctt ggg ttt ttc ctg tgg ctg aaa 3062 Ile Ser Thr Ala LeuLeu Leu Leu Leu Gly Phe Phe Leu Trp Leu Lys 945 950 955 aag aga aag caaatt aaa gat ctg ggc agt gaa tta gtt cgc tac gat 3110 Lys Arg Lys Gln IleLys Asp Leu Gly Ser Glu Leu Val Arg Tyr Asp 960 965 970 gca aga gta cacact cct cat ttg gat agg ctt gta agt gcc cga agt 3158 Ala Arg Val His ThrPro His Leu Asp Arg Leu Val Ser Ala Arg Ser 975 980 985 gta agc cca actaca gaa atg gtt tca aat gaa tct gta gac tac cga 3206 Val Ser Pro Thr ThrGlu Met Val Ser Asn Glu Ser Val Asp Tyr Arg 990 995 1000 gct act ttt ccagaa gat cag ttt cct aat tca tct cag aac ggt 3251 Ala Thr Phe Pro Glu AspGln Phe Pro Asn Ser Ser Gln Asn Gly 1005 1010 1015 tca tgc cga caa gtgcag tat cct ctg aca gac atg tcc ccc atc 3296 Ser Cys Arg Gln Val Gln TyrPro Leu Thr Asp Met Ser Pro Ile 1020 1025 1030 cta act agt ggg gac tctgat ata tcc agt cca tta ctg caa aat 3341 Leu Thr Ser Gly Asp Ser Asp IleSer Ser Pro Leu Leu Gln Asn 1035 1040 1045 act gtc cac att gac ctc agtgct cta aat cca gag ctg gtc cag 3386 Thr Val His Ile Asp Leu Ser Ala LeuAsn Pro Glu Leu Val Gln 1050 1055 1060 gca gtg cag cat gta gtg att gggccc agt agc ctg att gtg cat 3431 Ala Val Gln His Val Val Ile Gly Pro SerSer Leu Ile Val His 1065 1070 1075 ttc aat gaa gtc ata gga aga ggg catttt ggt tgt gta tat cat 3476 Phe Asn Glu Val Ile Gly Arg Gly His Phe GlyCys Val Tyr His 1080 1085 1090 ggg act ttg ttg gac aat gat ggc aag aaaatt cac tgt gct gtg 3521 Gly Thr Leu Leu Asp Asn Asp Gly Lys Lys Ile HisCys Ala Val 1095 1100 1105 aaa tcc ttg aac aga atc act gac ata gga gaagtt tcc caa ttt 3566 Lys Ser Leu Asn Arg Ile Thr Asp Ile Gly Glu Val SerGln Phe 1110 1115 1120 ctg acc gag gga atc atc atg aaa gat ttt agt catccc aat gtc 3611 Leu Thr Glu Gly Ile Ile Met Lys Asp Phe Ser His Pro AsnVal 1125 1130 1135 ctc tcg ctc ctg gga atc tgc ctg cga agt gaa ggg tctccg ctg 3656 Leu Ser Leu Leu Gly Ile Cys Leu Arg Ser Glu Gly Ser Pro Leu1140 1145 1150 gtg gtc cta cca tac atg aaa cat gga gat ctt cga aat ttcatt 3701 Val Val Leu Pro Tyr Met Lys His Gly Asp Leu Arg Asn Phe Ile1155 1160 1165 cga aat gag act cat aat cca act gta aaa gat ctt att ggcttt 3746 Arg Asn Glu Thr His Asn Pro Thr Val Lys Asp Leu Ile Gly Phe1170 1175 1180 ggt ctt caa gta gcc aaa ggc atg aaa tat ctt gca agc aaaaag 3791 Gly Leu Gln Val Ala Lys Gly Met Lys Tyr Leu Ala Ser Lys Lys1185 1190 1195 ttt gtc cac aga gac ttg gct gca aga aac tgt atg ctg gatgaa 3836 Phe Val His Arg Asp Leu Ala Ala Arg Asn Cys Met Leu Asp Glu1200 1205 1210 aaa ttc aca gtc aag gtt gct gat ttt ggt ctt gcc aga gacatg 3881 Lys Phe Thr Val Lys Val Ala Asp Phe Gly Leu Ala Arg Asp Met1215 1220 1225 tat gat aaa gaa tac tat agt gta cac aac aaa aca ggt gcaaag 3926 Tyr Asp Lys Glu Tyr Tyr Ser Val His Asn Lys Thr Gly Ala Lys1230 1235 1240 ctg cca gtg aag tgg atg gct ttg gaa agt ctg caa act caaaag 3971 Leu Pro Val Lys Trp Met Ala Leu Glu Ser Leu Gln Thr Gln Lys1245 1250 1255 ttt acc acc aag tca gat gtg tgg tcc ttt ggc gtc gtc ctctgg 4016 Phe Thr Thr Lys Ser Asp Val Trp Ser Phe Gly Val Val Leu Trp1260 1265 1270 gag ctg atg aca aga gga gcc cca cct tat cct gac gta aacacc 4061 Glu Leu Met Thr Arg Gly Ala Pro Pro Tyr Pro Asp Val Asn Thr1275 1280 1285 ttt gat ata act gtt tac ttg ttg caa ggg aga aga ctc ctacaa 4106 Phe Asp Ile Thr Val Tyr Leu Leu Gln Gly Arg Arg Leu Leu Gln1290 1295 1300 ccc gaa tac tgc cca gac ccc tta tat gaa gta atg cta aaatgc 4151 Pro Glu Tyr Cys Pro Asp Pro Leu Tyr Glu Val Met Leu Lys Cys1305 1310 1315 tgg cac cct aaa gcc gaa atg cgc cca tcc ttt tct gaa ctggtg 4196 Trp His Pro Lys Ala Glu Met Arg Pro Ser Phe Ser Glu Leu Val1320 1325 1330 tcc cgg ata tca gcg atc ttc tct act ttc att ggg gag cactat 4241 Ser Arg Ile Ser Ala Ile Phe Ser Thr Phe Ile Gly Glu His Tyr1335 1340 1345 gtc cat gtg aac gct act tat gtg aac gta aaa tgt gtc gctccg 4286 Val His Val Asn Ala Thr Tyr Val Asn Val Lys Cys Val Ala Pro1350 1355 1360 tat cct tct ctg ttg tca tca gaa gat aac gct gat gat gaggtg 4331 Tyr Pro Ser Leu Leu Ser Ser Glu Asp Asn Ala Asp Asp Glu Val1365 1370 1375 gac aca cga cca gcc tcc ttc tgg gag aca tca tagtgctagtact 4377 Asp Thr Arg Pro Ala Ser Phe Trp Glu Thr Ser 1380 13851390 atgtcaaagc aacagtccac actttgtcca atggtttttt cactgcctga cctttaaaag4437 gccatcgata ttctttgctc cttgccaaat tgcactatta ataggacttg tattgttatt4497 taaattactg gattctaagg aatttcttat ctgacagagc atcagaacca gaggcttggt4557 cccacaggcc agggaccaat gcgctgcag 4586 12 1390 PRT Homo sapiensSIGNAL (1)..(24) 12 Met Lys Ala Pro Ala Val Leu Ala Pro Gly Ile Leu ValLeu Leu Phe 1 5 10 15 Thr Leu Val Gln Arg Ser Asn Gly Glu Cys Lys GluAla Leu Ala Lys 20 25 30 Ser Glu Met Asn Val Asn Met Lys Tyr Gln Leu ProAsn Phe Thr Ala 35 40 45 Glu Thr Pro Ile Gln Asn Val Ile Leu His Glu HisHis Ile Phe Leu 50 55 60 Gly Ala Thr Asn Tyr Ile Tyr Val Leu Asn Glu GluAsp Leu Gln Lys 65 70 75 80 Val Ala Glu Tyr Lys Thr Gly Pro Val Leu GluHis Pro Asp Cys Phe 85 90 95 Pro Cys Gln Asp Cys Ser Ser Lys Ala Asn LeuSer Gly Gly Val Trp 100 105 110 Lys Asp Asn Ile Asn Met Ala Leu Val ValAsp Thr Tyr Tyr Asp Asp 115 120 125 Gln Leu Ile Ser Cys Gly Ser Val AsnArg Gly Thr Cys Gln Arg His 130 135 140 Val Phe Pro His Asn His Thr AlaAsp Ile Gln Ser Glu Val His Cys 145 150 155 160 Ile Phe Ser Pro Gln IleGlu Glu Pro Ser Gln Cys Pro Asp Cys Val 165 170 175 Val Ser Ala Leu GlyAla Lys Val Leu Ser Ser Val Lys Asp Arg Phe 180 185 190 Ile Asn Phe PheVal Gly Asn Thr Ile Asn Ser Ser Tyr Phe Pro Asp 195 200 205 His Pro LeuHis Ser Ile Ser Val Arg Arg Leu Lys Glu Thr Lys Asp 210 215 220 Gly PheMet Phe Leu Thr Asp Gln Ser Tyr Ile Asp Val Leu Pro Glu 225 230 235 240Phe Arg Asp Ser Tyr Pro Ile Lys Tyr Val His Ala Phe Glu Ser Asn 245 250255 Asn Phe Ile Tyr Phe Leu Thr Val Gln Arg Glu Thr Leu Asp Ala Gln 260265 270 Thr Phe His Thr Arg Ile Ile Arg Phe Cys Ser Ile Asn Ser Gly Leu275 280 285 His Ser Tyr Met Glu Met Pro Leu Glu Cys Ile Leu Thr Glu LysArg 290 295 300 Lys Lys Arg Ser Thr Lys Lys Glu Val Phe Asn Ile Leu GlnAla Ala 305 310 315 320 Tyr Val Ser Lys Pro Gly Ala Gln Leu Ala Arg GlnIle Gly Ala Ser 325 330 335 Leu Asn Asp Asp Ile Leu Phe Gly Val Phe AlaGln Ser Lys Pro Asp 340 345 350 Ser Ala Glu Pro Met Asp Arg Ser Ala MetCys Ala Phe Pro Ile Lys 355 360 365 Tyr Val Asn Asp Phe Phe Asn Lys IleVal Asn Lys Asn Asn Val Arg 370 375 380 Cys Leu Gln His Phe Tyr Gly ProAsn His Glu His Cys Phe Asn Arg 385 390 395 400 Thr Leu Leu Arg Asn SerSer Gly Cys Glu Ala Arg Arg Asp Glu Tyr 405 410 415 Arg Thr Glu Phe ThrThr Ala Leu Gln Arg Val Asp Leu Phe Met Gly 420 425 430 Gln Phe Ser GluVal Leu Leu Thr Ser Ile Ser Thr Phe Ile Lys Gly 435 440 445 Asp Leu ThrIle Ala Asn Leu Gly Thr Ser Glu Gly Arg Phe Met Gln 450 455 460 Val ValVal Ser Arg Ser Gly Pro Ser Thr Pro His Val Asn Phe Leu 465 470 475 480Leu Asp Ser His Pro Val Ser Pro Glu Val Ile Val Glu His Thr Leu 485 490495 Asn Gln Asn Gly Tyr Thr Leu Val Ile Thr Gly Lys Lys Ile Thr Lys 500505 510 Ile Pro Leu Asn Gly Leu Gly Cys Arg His Phe Gln Ser Cys Ser Gln515 520 525 Cys Leu Ser Ala Pro Pro Phe Val Gln Cys Gly Trp Cys His AspLys 530 535 540 Cys Val Arg Ser Glu Glu Cys Leu Ser Gly Thr Trp Thr GlnGln Ile 545 550 555 560 Cys Leu Pro Ala Ile Tyr Lys Val Phe Pro Asn SerAla Pro Leu Glu 565 570 575 Gly Gly Thr Arg Leu Thr Ile Cys Gly Trp AspPhe Gly Phe Arg Arg 580 585 590 Asn Asn Lys Phe Asp Leu Lys Lys Thr ArgVal Leu Leu Gly Asn Glu 595 600 605 Ser Cys Thr Leu Thr Leu Ser Glu SerThr Met Asn Thr Leu Lys Cys 610 615 620 Thr Val Gly Pro Ala Met Asn LysHis Phe Asn Met Ser Ile Ile Ile 625 630 635 640 Ser Asn Gly His Gly ThrThr Gln Tyr Ser Thr Phe Ser Tyr Val Asp 645 650 655 Pro Val Ile Thr SerIle Ser Pro Lys Tyr Gly Pro Met Ala Gly Gly 660 665 670 Thr Leu Leu ThrLeu Thr Gly Asn Tyr Leu Asn Ser Gly Asn Ser Arg 675 680 685 His Ile SerIle Gly Gly Lys Thr Cys Thr Leu Lys Ser Val Ser Asn 690 695 700 Ser IleLeu Glu Cys Tyr Thr Pro Ala Gln Thr Ile Ser Thr Glu Phe 705 710 715 720Ala Val Lys Leu Lys Ile Asp Leu Ala Asn Arg Glu Thr Ser Ile Phe 725 730735 Ser Tyr Arg Glu Asp Pro Ile Val Tyr Glu Ile His Pro Thr Lys Ser 740745 750 Phe Ile Ser Gly Gly Ser Thr Ile Thr Gly Val Gly Lys Asn Leu Asn755 760 765 Ser Val Ser Val Pro Arg Met Val Ile Asn Val His Glu Ala GlyArg 770 775 780 Asn Phe Thr Val Ala Cys Gln His Arg Ser Asn Ser Glu IleIle Cys 785 790 795 800 Cys Thr Thr Pro Ser Leu Gln Gln Leu Asn Leu GlnLeu Pro Leu Lys 805 810 815 Thr Lys Ala Phe Phe Met Leu Asp Gly Ile LeuSer Lys Tyr Phe Asp 820 825 830 Leu Ile Tyr Val His Asn Pro Val Phe LysPro Phe Glu Lys Pro Val 835 840 845 Met Ile Ser Met Gly Asn Glu Asn ValLeu Glu Ile Lys Gly Asn Asp 850 855 860 Ile Asp Pro Glu Ala Val Lys GlyGlu Val Leu Lys Val Gly Asn Lys 865 870 875 880 Ser Cys Glu Asn Ile HisLeu His Ser Glu Ala Val Leu Cys Thr Val 885 890 895 Pro Asn Asp Leu LeuLys Leu Asn Ser Glu Leu Asn Ile Glu Trp Lys 900 905 910 Gln Ala Ile SerSer Thr Val Leu Gly Lys Val Ile Val Gln Pro Asp 915 920 925 Gln Asn PheThr Gly Leu Ile Ala Gly Val Val Ser Ile Ser Thr Ala 930 935 940 Leu LeuLeu Leu Leu Gly Phe Phe Leu Trp Leu Lys Lys Arg Lys Gln 945 950 955 960Ile Lys Asp Leu Gly Ser Glu Leu Val Arg Tyr Asp Ala Arg Val His 965 970975 Thr Pro His Leu Asp Arg Leu Val Ser Ala Arg Ser Val Ser Pro Thr 980985 990 Thr Glu Met Val Ser Asn Glu Ser Val Asp Tyr Arg Ala Thr Phe Pro995 1000 1005 Glu Asp Gln Phe Pro Asn Ser Ser Gln Asn Gly Ser Cys ArgGln 1010 1015 1020 Val Gln Tyr Pro Leu Thr Asp Met Ser Pro Ile Leu ThrSer Gly 1025 1030 1035 Asp Ser Asp Ile Ser Ser Pro Leu Leu Gln Asn ThrVal His Ile 1040 1045 1050 Asp Leu Ser Ala Leu Asn Pro Glu Leu Val GlnAla Val Gln His 1055 1060 1065 Val Val Ile Gly Pro Ser Ser Leu Ile ValHis Phe Asn Glu Val 1070 1075 1080 Ile Gly Arg Gly His Phe Gly Cys ValTyr His Gly Thr Leu Leu 1085 1090 1095 Asp Asn Asp Gly Lys Lys Ile HisCys Ala Val Lys Ser Leu Asn 1100 1105 1110 Arg Ile Thr Asp Ile Gly GluVal Ser Gln Phe Leu Thr Glu Gly 1115 1120 1125 Ile Ile Met Lys Asp PheSer His Pro Asn Val Leu Ser Leu Leu 1130 1135 1140 Gly Ile Cys Leu ArgSer Glu Gly Ser Pro Leu Val Val Leu Pro 1145 1150 1155 Tyr Met Lys HisGly Asp Leu Arg Asn Phe Ile Arg Asn Glu Thr 1160 1165 1170 His Asn ProThr Val Lys Asp Leu Ile Gly Phe Gly Leu Gln Val 1175 1180 1185 Ala LysGly Met Lys Tyr Leu Ala Ser Lys Lys Phe Val His Arg 1190 1195 1200 AspLeu Ala Ala Arg Asn Cys Met Leu Asp Glu Lys Phe Thr Val 1205 1210 1215Lys Val Ala Asp Phe Gly Leu Ala Arg Asp Met Tyr Asp Lys Glu 1220 12251230 Tyr Tyr Ser Val His Asn Lys Thr Gly Ala Lys Leu Pro Val Lys 12351240 1245 Trp Met Ala Leu Glu Ser Leu Gln Thr Gln Lys Phe Thr Thr Lys1250 1255 1260 Ser Asp Val Trp Ser Phe Gly Val Val Leu Trp Glu Leu MetThr 1265 1270 1275 Arg Gly Ala Pro Pro Tyr Pro Asp Val Asn Thr Phe AspIle Thr 1280 1285 1290 Val Tyr Leu Leu Gln Gly Arg Arg Leu Leu Gln ProGlu Tyr Cys 1295 1300 1305 Pro Asp Pro Leu Tyr Glu Val Met Leu Lys CysTrp His Pro Lys 1310 1315 1320 Ala Glu Met Arg Pro Ser Phe Ser Glu LeuVal Ser Arg Ile Ser 1325 1330 1335 Ala Ile Phe Ser Thr Phe Ile Gly GluHis Tyr Val His Val Asn 1340 1345 1350 Ala Thr Tyr Val Asn Val Lys CysVal Ala Pro Tyr Pro Ser Leu 1355 1360 1365 Leu Ser Ser Glu Asp Asn AlaAsp Asp Glu Val Asp Thr Arg Pro 1370 1375 1380 Ala Ser Phe Trp Glu ThrSer 1385 1390 13 2558 DNA Homo sapiens gene (1)..(2558) Chondroitinsulfate proteoglycan BEHAB/brevican mRNA, GPI isoform, complete cds 13tgtggcactg cctgcgtacc caaccccagc cctgggtagc ctgcagc atg gcc cag 56 MetAla Gln 1 ctg ttc ctg ccc ctg ctg gca gcc ctg gtc ctg gcc cag gct cctgca 104 Leu Phe Leu Pro Leu Leu Ala Ala Leu Val Leu Ala Gln Ala Pro Ala5 10 15 gct tta gca gat gtt ctg gaa gga gac agc tca gag gac cgc gct ttt152 Ala Leu Ala Asp Val Leu Glu Gly Asp Ser Ser Glu Asp Arg Ala Phe 2025 30 35 cgc gtg cgc atc gcg ggc gac gcg cca ctg cag ggc gtg ctc ggc ggc200 Arg Val Arg Ile Ala Gly Asp Ala Pro Leu Gln Gly Val Leu Gly Gly 4045 50 gcc ctc acc atc cct tgc cac gtc cac tac ctg cgg cca ccg ccg agc248 Ala Leu Thr Ile Pro Cys His Val His Tyr Leu Arg Pro Pro Pro Ser 5560 65 cgc cgg gct gtg ctg ggc tct ccg cgg gtc aag tgg act ttc ctg tcc296 Arg Arg Ala Val Leu Gly Ser Pro Arg Val Lys Trp Thr Phe Leu Ser 7075 80 cgg ggc cgg gag gca gag gtg ctg gtg gcg cgg gga gtg cgc gtc aag344 Arg Gly Arg Glu Ala Glu Val Leu Val Ala Arg Gly Val Arg Val Lys 8590 95 gtg aac gag gcc tac cgg ttc cgc gtg gca ctg cct gcg tac cca gcg392 Val Asn Glu Ala Tyr Arg Phe Arg Val Ala Leu Pro Ala Tyr Pro Ala 100105 110 115 tcg ctc acc gac gtc tcc ctg gcg ctg agc gag ctg cgc ccc aacgac 440 Ser Leu Thr Asp Val Ser Leu Ala Leu Ser Glu Leu Arg Pro Asn Asp120 125 130 tca ggt atc tat cgc tgt gag gtc cag cac ggc atc gat gac agcagc 488 Ser Gly Ile Tyr Arg Cys Glu Val Gln His Gly Ile Asp Asp Ser Ser135 140 145 gac gct gtg gag gtc aag gtc aaa ggg gtc gtc ttt ctc tac cgagag 536 Asp Ala Val Glu Val Lys Val Lys Gly Val Val Phe Leu Tyr Arg Glu150 155 160 ggc tct gcc cgc tat gct ttc tcc ttt tct ggg gcc cag gag gcctgt 584 Gly Ser Ala Arg Tyr Ala Phe Ser Phe Ser Gly Ala Gln Glu Ala Cys165 170 175 gcc cgc att gga gcc cac atc gcc acc ccg gag cag ctc tat gccgcc 632 Ala Arg Ile Gly Ala His Ile Ala Thr Pro Glu Gln Leu Tyr Ala Ala180 185 190 195 tac ctt ggg ggc tat gag caa tgt gat gct ggc tgg ctg tcggat cag 680 Tyr Leu Gly Gly Tyr Glu Gln Cys Asp Ala Gly Trp Leu Ser AspGln 200 205 210 acc gtg agg tat ccc atc cag acc cca cga gag gcc tgt tacgga gac 728 Thr Val Arg Tyr Pro Ile Gln Thr Pro Arg Glu Ala Cys Tyr GlyAsp 215 220 225 atg gat ggc ttc ccc ggg gtc cgg aac tat ggt gtg gtg gacccg gat 776 Met Asp Gly Phe Pro Gly Val Arg Asn Tyr Gly Val Val Asp ProAsp 230 235 240 gac ctc tat gat gtg tac tgt tat gct gaa gac cta aat ggagaa ttg 824 Asp Leu Tyr Asp Val Tyr Cys Tyr Ala Glu Asp Leu Asn Gly GluLeu 245 250 255 ttc ctg ggt gac cct cca gag aag ctg aca ttg gag gaa gcacgg gcg 872 Phe Leu Gly Asp Pro Pro Glu Lys Leu Thr Leu Glu Glu Ala ArgAla 260 265 270 275 tac tgc cag gag cgg ggt gca gag att gcc acc acg ggccaa ctg tat 920 Tyr Cys Gln Glu Arg Gly Ala Glu Ile Ala Thr Thr Gly GlnLeu Tyr 280 285 290 gca gcc tgg gat ggt ggc ctg gac cac tgc agc cca gggtgg cta gct 968 Ala Ala Trp Asp Gly Gly Leu Asp His Cys Ser Pro Gly TrpLeu Ala 295 300 305 gat ggc agt gtg cgc tac ccc atc gtc aca ccc agc cagcgc tgt ggt 1016 Asp Gly Ser Val Arg Tyr Pro Ile Val Thr Pro Ser Gln ArgCys Gly 310 315 320 ggg ggc ttg cct ggt gtc aag act ctc ttc ctc ttc cccaac cag act 1064 Gly Gly Leu Pro Gly Val Lys Thr Leu Phe Leu Phe Pro AsnGln Thr 325 330 335 ggc ttc ccc aat aag cac agc cgc ttc aac gtc tac tgcttc cga gac 1112 Gly Phe Pro Asn Lys His Ser Arg Phe Asn Val Tyr Cys PheArg Asp 340 345 350 355 tcg gcc cag cct tct gcc atc cct gag gcc tcc aaccca gcc tcc aac 1160 Ser Ala Gln Pro Ser Ala Ile Pro Glu Ala Ser Asn ProAla Ser Asn 360 365 370 cca gcc tct gat gga cta gag gct atc gtc aca gtgaca gag acc ctg 1208 Pro Ala Ser Asp Gly Leu Glu Ala Ile Val Thr Val ThrGlu Thr Leu 375 380 385 gag gaa ctg cag ctg cct cag gaa gcc aca gag agtgaa tcc cgt ggg 1256 Glu Glu Leu Gln Leu Pro Gln Glu Ala Thr Glu Ser GluSer Arg Gly 390 395 400 gcc atc tac tcc atc ccc atc atg gag gac gga ggaggt gga agc tcc 1304 Ala Ile Tyr Ser Ile Pro Ile Met Glu Asp Gly Gly GlyGly Ser Ser 405 410 415 act cca gaa gac cca gca gag gcc cct agg acg ctccta gaa ttt gaa 1352 Thr Pro Glu Asp Pro Ala Glu Ala Pro Arg Thr Leu LeuGlu Phe Glu 420 425 430 435 aca caa tcc atg gta ccg ccc acg ggg ttc tcagaa gag gaa ggt aag 1400 Thr Gln Ser Met Val Pro Pro Thr Gly Phe Ser GluGlu Glu Gly Lys 440 445 450 gca ttg gag gaa gaa gag aaa tat gaa gat gaagaa gag aaa gag gag 1448 Ala Leu Glu Glu Glu Glu Lys Tyr Glu Asp Glu GluGlu Lys Glu Glu 455 460 465 gaa gaa gaa gag gag gag gtg gag gat gag gctctg tgg gca tgg ccc 1496 Glu Glu Glu Glu Glu Glu Val Glu Asp Glu Ala LeuTrp Ala Trp Pro 470 475 480 agc gag ctc agc agc ccg ggc cct gag gcc tctctc ccc act gag cca 1544 Ser Glu Leu Ser Ser Pro Gly Pro Glu Ala Ser LeuPro Thr Glu Pro 485 490 495 gca gcc cag gag gag tca ctc tcc cag gcg ccagca agg gca gtc ctg 1592 Ala Ala Gln Glu Glu Ser Leu Ser Gln Ala Pro AlaArg Ala Val Leu 500 505 510 515 cag cct ggt gca tca cca ctt cct gat ggagag tca gaa gct tcc agg 1640 Gln Pro Gly Ala Ser Pro Leu Pro Asp Gly GluSer Glu Ala Ser Arg 520 525 530 cct cca agg gtc cat gga cca cct act gagact ctg ccc act ccc agg 1688 Pro Pro Arg Val His Gly Pro Pro Thr Glu ThrLeu Pro Thr Pro Arg 535 540 545 gag agg aac cta gca tcc cca tca cct tccact ctg gtt gag gca aga 1736 Glu Arg Asn Leu Ala Ser Pro Ser Pro Ser ThrLeu Val Glu Ala Arg 550 555 560 gag gtg ggg gag gca act ggt ggt cct gagcta tct ggg gtc cct cga 1784 Glu Val Gly Glu Ala Thr Gly Gly Pro Glu LeuSer Gly Val Pro Arg 565 570 575 gga gag agc gag gag aca gga agc tcc gagggt gcc cct tcc ctg ctt 1832 Gly Glu Ser Glu Glu Thr Gly Ser Ser Glu GlyAla Pro Ser Leu Leu 580 585 590 595 cca gcc aca cgg gcc cct gag ggt accagg gag ctg gag gcc ccc tct 1880 Pro Ala Thr Arg Ala Pro Glu Gly Thr ArgGlu Leu Glu Ala Pro Ser 600 605 610 gaa gat aat tct gga aga act gcc ccagca ggg acc tca gtg cag gcc 1928 Glu Asp Asn Ser Gly Arg Thr Ala Pro AlaGly Thr Ser Val Gln Ala 615 620 625 cag cca gtg ctg ccc act gac agc gccagc cga ggt gga gtg gcc gtg 1976 Gln Pro Val Leu Pro Thr Asp Ser Ala SerArg Gly Gly Val Ala Val 630 635 640 gtc ccc gca tca ggt aat tct gcc caaggc tca act gcc ctc tct atc 2024 Val Pro Ala Ser Gly Asn Ser Ala Gln GlySer Thr Ala Leu Ser Ile 645 650 655 cta ctc ctt ttc ttc ccc ctg cag ctctgg gtc acc tga cctgtagtcc 2073 Leu Leu Leu Phe Phe Pro Leu Gln Leu TrpVal Thr 660 665 670 tttaacccac catcatccca aactctcctg tcctttgccttcattctctt acccacctct 2133 acctatgggt ctccaatctc ggatatccac cttgtgggtatctcagctct ccgcgtcttt 2193 accctgtgat cccagccccg ccactgacca tctgtgacccttccctgcca ttgggccctc 2253 cacctgtggc tcacatctcg ccagccccac agagcatcctcaggcctctc caagggtcct 2313 catcacctat tgcagccttc agggctcggc ctattttccactactccctt catccgcctg 2373 tgtgccgtcc cctttagctg cctcctattg atctcagggaagcctgggag tcccttctca 2433 cccctcaacc tccggagtcc aggagaaccc gtacccccacagagccttaa gcaactactt 2493 ctgtgaagta ttttttgact gtttcatgga aaacaagccttggaaataaa tctctattaa 2553 accgc 2558 14 671 PRT Homo sapiens gene(1)..(671) Chondroitin sulfate proteoglycan BEHAB/brevican 14 Met AlaGln Leu Phe Leu Pro Leu Leu Ala Ala Leu Val Leu Ala Gln 1 5 10 15 AlaPro Ala Ala Leu Ala Asp Val Leu Glu Gly Asp Ser Ser Glu Asp 20 25 30 ArgAla Phe Arg Val Arg Ile Ala Gly Asp Ala Pro Leu Gln Gly Val 35 40 45 LeuGly Gly Ala Leu Thr Ile Pro Cys His Val His Tyr Leu Arg Pro 50 55 60 ProPro Ser Arg Arg Ala Val Leu Gly Ser Pro Arg Val Lys Trp Thr 65 70 75 80Phe Leu Ser Arg Gly Arg Glu Ala Glu Val Leu Val Ala Arg Gly Val 85 90 95Arg Val Lys Val Asn Glu Ala Tyr Arg Phe Arg Val Ala Leu Pro Ala 100 105110 Tyr Pro Ala Ser Leu Thr Asp Val Ser Leu Ala Leu Ser Glu Leu Arg 115120 125 Pro Asn Asp Ser Gly Ile Tyr Arg Cys Glu Val Gln His Gly Ile Asp130 135 140 Asp Ser Ser Asp Ala Val Glu Val Lys Val Lys Gly Val Val PheLeu 145 150 155 160 Tyr Arg Glu Gly Ser Ala Arg Tyr Ala Phe Ser Phe SerGly Ala Gln 165 170 175 Glu Ala Cys Ala Arg Ile Gly Ala His Ile Ala ThrPro Glu Gln Leu 180 185 190 Tyr Ala Ala Tyr Leu Gly Gly Tyr Glu Gln CysAsp Ala Gly Trp Leu 195 200 205 Ser Asp Gln Thr Val Arg Tyr Pro Ile GlnThr Pro Arg Glu Ala Cys 210 215 220 Tyr Gly Asp Met Asp Gly Phe Pro GlyVal Arg Asn Tyr Gly Val Val 225 230 235 240 Asp Pro Asp Asp Leu Tyr AspVal Tyr Cys Tyr Ala Glu Asp Leu Asn 245 250 255 Gly Glu Leu Phe Leu GlyAsp Pro Pro Glu Lys Leu Thr Leu Glu Glu 260 265 270 Ala Arg Ala Tyr CysGln Glu Arg Gly Ala Glu Ile Ala Thr Thr Gly 275 280 285 Gln Leu Tyr AlaAla Trp Asp Gly Gly Leu Asp His Cys Ser Pro Gly 290 295 300 Trp Leu AlaAsp Gly Ser Val Arg Tyr Pro Ile Val Thr Pro Ser Gln 305 310 315 320 ArgCys Gly Gly Gly Leu Pro Gly Val Lys Thr Leu Phe Leu Phe Pro 325 330 335Asn Gln Thr Gly Phe Pro Asn Lys His Ser Arg Phe Asn Val Tyr Cys 340 345350 Phe Arg Asp Ser Ala Gln Pro Ser Ala Ile Pro Glu Ala Ser Asn Pro 355360 365 Ala Ser Asn Pro Ala Ser Asp Gly Leu Glu Ala Ile Val Thr Val Thr370 375 380 Glu Thr Leu Glu Glu Leu Gln Leu Pro Gln Glu Ala Thr Glu SerGlu 385 390 395 400 Ser Arg Gly Ala Ile Tyr Ser Ile Pro Ile Met Glu AspGly Gly Gly 405 410 415 Gly Ser Ser Thr Pro Glu Asp Pro Ala Glu Ala ProArg Thr Leu Leu 420 425 430 Glu Phe Glu Thr Gln Ser Met Val Pro Pro ThrGly Phe Ser Glu Glu 435 440 445 Glu Gly Lys Ala Leu Glu Glu Glu Glu LysTyr Glu Asp Glu Glu Glu 450 455 460 Lys Glu Glu Glu Glu Glu Glu Glu GluVal Glu Asp Glu Ala Leu Trp 465 470 475 480 Ala Trp Pro Ser Glu Leu SerSer Pro Gly Pro Glu Ala Ser Leu Pro 485 490 495 Thr Glu Pro Ala Ala GlnGlu Glu Ser Leu Ser Gln Ala Pro Ala Arg 500 505 510 Ala Val Leu Gln ProGly Ala Ser Pro Leu Pro Asp Gly Glu Ser Glu 515 520 525 Ala Ser Arg ProPro Arg Val His Gly Pro Pro Thr Glu Thr Leu Pro 530 535 540 Thr Pro ArgGlu Arg Asn Leu Ala Ser Pro Ser Pro Ser Thr Leu Val 545 550 555 560 GluAla Arg Glu Val Gly Glu Ala Thr Gly Gly Pro Glu Leu Ser Gly 565 570 575Val Pro Arg Gly Glu Ser Glu Glu Thr Gly Ser Ser Glu Gly Ala Pro 580 585590 Ser Leu Leu Pro Ala Thr Arg Ala Pro Glu Gly Thr Arg Glu Leu Glu 595600 605 Ala Pro Ser Glu Asp Asn Ser Gly Arg Thr Ala Pro Ala Gly Thr Ser610 615 620 Val Gln Ala Gln Pro Val Leu Pro Thr Asp Ser Ala Ser Arg GlyGly 625 630 635 640 Val Ala Val Val Pro Ala Ser Gly Asn Ser Ala Gln GlySer Thr Ala 645 650 655 Leu Ser Ile Leu Leu Leu Phe Phe Pro Leu Gln LeuTrp Val Thr 660 665 670 15 2316 DNA Homo sapiens gene (1)..(2316) HumanmRNA for CD44E (epithelial form) 15 agcggacccc agcctctgcc aggttcggtccgccatcctc gtcccgtcct ccgccggccc 60 ctgccccgcg cccagggatc ctccagctcctttcgcccgc gccctccgtt cgctccggac 120 acc atg gac aag ttt tgg tgg cac gcagcc tgg gga ctc tgc ctc gtg 168 Met Asp Lys Phe Trp Trp His Ala Ala TrpGly Leu Cys Leu Val 1 5 10 15 ccg ctg agc ctg gcg cag atc gat ttg aatata acc tgc cgc ttt gca 216 Pro Leu Ser Leu Ala Gln Ile Asp Leu Asn IleThr Cys Arg Phe Ala 20 25 30 ggt gta ttc cac gtg gag aaa aat ggt cgc tacagc atc tct cgg acg 264 Gly Val Phe His Val Glu Lys Asn Gly Arg Tyr SerIle Ser Arg Thr 35 40 45 gag gcc gct gac ctc tgc aag gct ttc aat agc accttg ccc aca atg 312 Glu Ala Ala Asp Leu Cys Lys Ala Phe Asn Ser Thr LeuPro Thr Met 50 55 60 gcc cag atg gag aaa gct ctg agc atc gga ttt gag acctgc agg tat 360 Ala Gln Met Glu Lys Ala Leu Ser Ile Gly Phe Glu Thr CysArg Tyr 65 70 75 ggg ttc ata gaa ggg cat gtg gtg att ccc cgg atc cac cccaac tcc 408 Gly Phe Ile Glu Gly His Val Val Ile Pro Arg Ile His Pro AsnSer 80 85 90 95 atc tgt gca gca aac aac aca ggg gtg tac atc ctc aca tacaac acc 456 Ile Cys Ala Ala Asn Asn Thr Gly Val Tyr Ile Leu Thr Tyr AsnThr 100 105 110 tcc cag tat gac aca tat tgc ttc aat gct tca gct cca cctgaa gaa 504 Ser Gln Tyr Asp Thr Tyr Cys Phe Asn Ala Ser Ala Pro Pro GluGlu 115 120 125 gat tgt aca tca gtc aca gac ctg ccc aat gcc ttt gat ggacca att 552 Asp Cys Thr Ser Val Thr Asp Leu Pro Asn Ala Phe Asp Gly ProIle 130 135 140 acc ata act att gtt aac cgt gat ggc acc cgc tat gtc cagaaa gga 600 Thr Ile Thr Ile Val Asn Arg Asp Gly Thr Arg Tyr Val Gln LysGly 145 150 155 gaa tac aga acg aat cct gaa gac atc tac ccc agc aac cctact gat 648 Glu Tyr Arg Thr Asn Pro Glu Asp Ile Tyr Pro Ser Asn Pro ThrAsp 160 165 170 175 gat gac gtg agc agc ggc tcc tcc agt gaa agg agc agcact tca gga 696 Asp Asp Val Ser Ser Gly Ser Ser Ser Glu Arg Ser Ser ThrSer Gly 180 185 190 ggt tac atc ttt tac acc ttt tct act gta cac ccc atccca gac gaa 744 Gly Tyr Ile Phe Tyr Thr Phe Ser Thr Val His Pro Ile ProAsp Glu 195 200 205 gac agt ccc tgg atc acc gac agc aca gac aga atc cctcgt acc aat 792 Asp Ser Pro Trp Ile Thr Asp Ser Thr Asp Arg Ile Pro ArgThr Asn 210 215 220 atg gac tcc agt cat agt aca acg ctt cag cct act gcaaat cca aac 840 Met Asp Ser Ser His Ser Thr Thr Leu Gln Pro Thr Ala AsnPro Asn 225 230 235 aca ggt ttg gtg gaa gat ttg gac agg aca gga cct ctttca atg aca 888 Thr Gly Leu Val Glu Asp Leu Asp Arg Thr Gly Pro Leu SerMet Thr 240 245 250 255 acg cag cag agt aat tct cag agc ttc tct aca tcacat gaa ggc ttg 936 Thr Gln Gln Ser Asn Ser Gln Ser Phe Ser Thr Ser HisGlu Gly Leu 260 265 270 gaa gaa gat aaa gac cat cca aca act tct act ctgaca tca agc aat 984 Glu Glu Asp Lys Asp His Pro Thr Thr Ser Thr Leu ThrSer Ser Asn 275 280 285 agg aat gat gtc aca ggt gga aga aga gac cca aatcat tct gaa ggc 1032 Arg Asn Asp Val Thr Gly Gly Arg Arg Asp Pro Asn HisSer Glu Gly 290 295 300 tca act cat tta ctg gaa ggt tat acc tct cat taccca cac acg aag 1080 Ser Thr His Leu Leu Glu Gly Tyr Thr Ser His Tyr ProHis Thr Lys 305 310 315 gaa agc agg acc ttc atc cca gtg acc tca gct aagact ggg tcc ttt 1128 Glu Ser Arg Thr Phe Ile Pro Val Thr Ser Ala Lys ThrGly Ser Phe 320 325 330 335 gga gtt act gca gtt act gtt gga gat tcc aactct aat gtc aat cgt 1176 Gly Val Thr Ala Val Thr Val Gly Asp Ser Asn SerAsn Val Asn Arg 340 345 350 tcc tta tca gga gac caa gac aca ttc cac cccagt ggg ggg tcc cat 1224 Ser Leu Ser Gly Asp Gln Asp Thr Phe His Pro SerGly Gly Ser His 355 360 365 acc act cat gga tct gaa tca gat gga cac tcacat ggg agt caa gaa 1272 Thr Thr His Gly Ser Glu Ser Asp Gly His Ser HisGly Ser Gln Glu 370 375 380 ggt gga gca aac aca acc tct ggt cct ata aggaca ccc caa att cca 1320 Gly Gly Ala Asn Thr Thr Ser Gly Pro Ile Arg ThrPro Gln Ile Pro 385 390 395 gaa tgg ctg atc atc ttg gca tcc ctc ttg gccttg gct ttg att ctt 1368 Glu Trp Leu Ile Ile Leu Ala Ser Leu Leu Ala LeuAla Leu Ile Leu 400 405 410 415 gca gtt tgc att gca gtc aac agt cga agaagg tgt ggg cag aag aaa 1416 Ala Val Cys Ile Ala Val Asn Ser Arg Arg ArgCys Gly Gln Lys Lys 420 425 430 aag cta gtg atc aac agt ggc aat gga gctgtg gag gac aga aag cca 1464 Lys Leu Val Ile Asn Ser Gly Asn Gly Ala ValGlu Asp Arg Lys Pro 435 440 445 agt gga ctc aac gga gag gcc agc aag tctcag gaa atg gtg cat ttg 1512 Ser Gly Leu Asn Gly Glu Ala Ser Lys Ser GlnGlu Met Val His Leu 450 455 460 gtg aac aag gag tcg tca gaa act cca gaccag ttt atg aca gct gat 1560 Val Asn Lys Glu Ser Ser Glu Thr Pro Asp GlnPhe Met Thr Ala Asp 465 470 475 gag aca agg aac ctg cag aat gtg gac atgaag att ggg gtg taa 1605 Glu Thr Arg Asn Leu Gln Asn Val Asp Met Lys IleGly Val 480 485 490 cacctacacc attatcttgg aaagaaacaa cgttggaaacataaccatta caggggagct 1665 gggacactta acagatgcaa tgtgctactg attgtttcatttcgaatcta taatagcata 1725 aaattttcta ctctttttgt tttttgtgtt ttgttctttaaagtcaggtc caatttgtaa 1785 aaacagcatt gctttctgaa attagggccc aattaataatcagcaagaat tttgatcgtt 1845 tcagttcccc acttggaggc ctttcatccc tcgggtgtgctatggatggc ttctaacaaa 1905 aacctaccac atagttattc ctgatcgcca accttgccccccaccagcta aggacatttc 1965 cagggttaat agggcctggt cctgggagga aatttgaatgggtcattttg cccttccatt 2025 agcctaatcc ctgggcattg ctttccactg aggttgggggttggggtgta ctagttacac 2085 atcttcaaca gaccccctct agaaattttt cagatgcttctgggagacac ccaaagggta 2145 agtctattta tctgtagtaa actatttatc tgtgtttttgaaatattaaa ccctggatca 2205 gtccttttat tcagtataat tttttaaagt tactttgtcagaggcacaaa aagggtttaa 2265 actgattcat aataaatatc tgtaccttct tcgaaaaaaaaaaaaaaaaa a 2316 16 742 PRT Homo sapiens SIGNAL (1)..(20) BY SIMILARITY16 Met Asp Lys Phe Trp Trp His Ala Ala Trp Gly Leu Cys Leu Val Pro 1 510 15 Leu Ser Leu Ala Gln Ile Asp Leu Asn Ile Thr Cys Arg Phe Ala Gly 2025 30 Val Phe His Val Glu Lys Asn Gly Arg Tyr Ser Ile Ser Arg Thr Glu 3540 45 Ala Ala Asp Leu Cys Lys Ala Phe Asn Ser Thr Leu Pro Thr Met Ala 5055 60 Gln Met Glu Lys Ala Leu Ser Ile Gly Phe Glu Thr Cys Arg Tyr Gly 6570 75 80 Phe Ile Glu Gly His Val Val Ile Pro Arg Ile His Pro Asn Ser Ile85 90 95 Cys Ala Ala Asn Asn Thr Gly Val Tyr Ile Leu Thr Ser Asn Thr Ser100 105 110 Gln Tyr Asp Thr Tyr Cys Phe Asn Ala Ser Ala Pro Pro Glu GluAsp 115 120 125 Cys Thr Ser Val Thr Asp Leu Pro Asn Ala Phe Asp Gly ProIle Thr 130 135 140 Ile Thr Ile Val Asn Arg Asp Gly Thr Arg Tyr Val GlnLys Gly Glu 145 150 155 160 Tyr Arg Thr Asn Pro Glu Asp Ile Tyr Pro SerAsn Pro Thr Asp Asp 165 170 175 Asp Val Ser Ser Gly Ser Ser Ser Glu ArgSer Ser Thr Ser Gly Gly 180 185 190 Tyr Ile Phe Tyr Thr Phe Ser Thr ValHis Pro Ile Pro Asp Glu Asp 195 200 205 Ser Pro Trp Ile Thr Asp Ser ThrAsp Arg Ile Pro Ala Thr Thr Leu 210 215 220 Met Ser Thr Ser Ala Thr AlaThr Glu Thr Ala Thr Lys Arg Gln Glu 225 230 235 240 Thr Trp Asp Trp PheSer Trp Leu Phe Leu Pro Ser Glu Ser Lys Asn 245 250 255 His Leu His ThrThr Thr Gln Met Ala Gly Thr Ser Ser Asn Thr Ile 260 265 270 Ser Ala GlyTrp Glu Pro Asn Glu Glu Asn Glu Asp Glu Arg Asp Arg 275 280 285 His LeuSer Phe Ser Gly Ser Gly Ile Asp Asp Asp Glu Asp Phe Ile 290 295 300 SerSer Thr Ile Ser Thr Thr Pro Arg Ala Phe Asp His Thr Lys Gln 305 310 315320 Asn Gln Asp Trp Thr Gln Trp Asn Pro Ser His Ser Asn Pro Glu Val 325330 335 Leu Leu Gln Thr Thr Thr Arg Met Thr Asp Val Asp Arg Asn Gly Thr340 345 350 Thr Ala Tyr Glu Gly Asn Trp Asn Pro Glu Ala His Pro Pro LeuIle 355 360 365 His His Glu His His Glu Glu Glu Glu Thr Pro His Ser ThrSer Thr 370 375 380 Ile Gln Ala Thr Pro Ser Ser Thr Thr Glu Glu Thr AlaThr Gln Lys 385 390 395 400 Glu Gln Trp Phe Gly Asn Arg Trp His Glu GlyTyr Arg Gln Thr Pro 405 410 415 Arg Glu Asp Ser His Ser Thr Thr Gly ThrAla Ala Ala Ser Ala His 420 425 430 Thr Ser His Pro Met Gln Gly Arg ThrThr Pro Ser Pro Glu Asp Ser 435 440 445 Ser Trp Thr Asp Phe Phe Asn ProIle Ser His Pro Met Gly Arg Gly 450 455 460 His Gln Ala Gly Arg Arg MetAsp Met Asp Ser Ser His Ser Thr Thr 465 470 475 480 Leu Gln Pro Thr AlaAsn Pro Asn Thr Gly Leu Val Glu Asp Leu Asp 485 490 495 Arg Thr Gly ProLeu Ser Met Thr Thr Gln Gln Ser Asn Ser Gln Ser 500 505 510 Phe Ser ThrSer His Glu Gly Leu Glu Glu Asp Lys Asp His Pro Thr 515 520 525 Thr SerThr Leu Thr Ser Ser Asn Arg Asn Asp Val Thr Gly Gly Arg 530 535 540 ArgAsp Pro Asn His Ser Glu Gly Ser Thr Thr Leu Leu Glu Gly Tyr 545 550 555560 Thr Ser His Tyr Pro His Thr Lys Glu Ser Arg Thr Phe Ile Pro Val 565570 575 Thr Ser Ala Lys Thr Gly Ser Phe Gly Val Thr Ala Val Thr Val Gly580 585 590 Asp Ser Asn Ser Asn Val Asn Arg Ser Leu Ser Gly Asp Gln AspThr 595 600 605 Phe His Pro Ser Gly Gly Ser His Thr Thr His Gly Ser GluSer Asp 610 615 620 Gly His Ser His Gly Ser Gln Glu Gly Gly Ala Asn ThrThr Ser Gly 625 630 635 640 Pro Ile Arg Thr Pro Gln Ile Pro Glu Trp LeuIle Ile Leu Ala Ser 645 650 655 Leu Leu Ala Leu Ala Leu Ile Leu Ala ValCys Ile Ala Val Asn Ser 660 665 670 Arg Arg Arg Cys Gly Gln Lys Lys LysLeu Val Ile Asn Ser Gly Asn 675 680 685 Gly Ala Val Glu Asp Arg Lys ProSer Gly Leu Asn Gly Glu Ala Ser 690 695 700 Lys Ser Gln Glu Met Val HisLeu Val Asn Lys Glu Ser Ser Glu Thr 705 710 715 720 Pro Asp Gln Phe MetThr Ala Asp Glu Thr Arg Asn Leu Gln Asn Val 725 730 735 Asp Met Lys IleGly Val 740 17 930 DNA Homo sapiens Gene (1)..(930) Tetraspan TM4SF(TSPAN-3), mRNA 17 atg ggc cag tgc ggc atc acc tcc tcc aag acc gtg ctggtc ttt ctc 48 Met Gly Gln Cys Gly Ile Thr Ser Ser Lys Thr Val Leu ValPhe Leu 1 5 10 15 aac ctc atc ttc tgg ggg gca gct ggc att tta tgc tatgtg gga gcc 96 Asn Leu Ile Phe Trp Gly Ala Ala Gly Ile Leu Cys Tyr ValGly Ala 20 25 30 tat gtc ttc atc act tat gat gac tat gac cac ttc ttt gaagat gtg 144 Tyr Val Phe Ile Thr Tyr Asp Asp Tyr Asp His Phe Phe Glu AspVal 35 40 45 tac acg ctc atc cct gct gta gtg atc ata gct gta gga gcc ctgctt 192 Tyr Thr Leu Ile Pro Ala Val Val Ile Ile Ala Val Gly Ala Leu Leu50 55 60 ttc atc att ggg cta att ggc tgc tgt gcc aca atc cgg gaa agt cgc240 Phe Ile Ile Gly Leu Ile Gly Cys Cys Ala Thr Ile Arg Glu Ser Arg 6570 75 80 tgt gga ctt gcc acg ttt gtc atc atc ctg ctc ttg gtt ttt gtc aca288 Cys Gly Leu Ala Thr Phe Val Ile Ile Leu Leu Leu Val Phe Val Thr 8590 95 gaa gtt gtt gta gtg gtt ttg gga tat gtt tac aga gca aag gtg gaa336 Glu Val Val Val Val Val Leu Gly Tyr Val Tyr Arg Ala Lys Val Glu 100105 110 aat gag gtt gat cgc agc att cag aaa gtg tat aag acc tac aat gga384 Asn Glu Val Asp Arg Ser Ile Gln Lys Val Tyr Lys Thr Tyr Asn Gly 115120 125 acc aac cct gat gct gct agc cgg gct att gat tat gta cag aga cag432 Thr Asn Pro Asp Ala Ala Ser Arg Ala Ile Asp Tyr Val Gln Arg Gln 130135 140 ctg cat tgt tgt gga att cac aac tac tca gac tgg gaa aat aca gat480 Leu His Cys Cys Gly Ile His Asn Tyr Ser Asp Trp Glu Asn Thr Asp 145150 155 160 tgg ttc aaa gaa acc aaa aac cag agt gtc cct ctt agc tgc tgcaga 528 Trp Phe Lys Glu Thr Lys Asn Gln Ser Val Pro Leu Ser Cys Cys Arg165 170 175 gag act gcc agc aat tgt aat ggc agc ctg gcc cac cct tcc gacctc 576 Glu Thr Ala Ser Asn Cys Asn Gly Ser Leu Ala His Pro Ser Asp Leu180 185 190 tat gct gag ggg tgt gag gct cta gtt gtg aag aag cta caa gaaatc 624 Tyr Ala Glu Gly Cys Glu Ala Leu Val Val Lys Lys Leu Gln Glu Ile195 200 205 atg atg cat gtg atc tgg gcc gca ctg gca ttt gca gct att cagctg 672 Met Met His Val Ile Trp Ala Ala Leu Ala Phe Ala Ala Ile Gln Leu210 215 220 ctg ggc atg ctg tgt gct tgc atc gtg ttg tgc aga agg agt agagat 720 Leu Gly Met Leu Cys Ala Cys Ile Val Leu Cys Arg Arg Ser Arg Asp225 230 235 240 cct gct tac gag ctc ctc atc act ggc gga acc tat gca tag762 Pro Ala Tyr Glu Leu Leu Ile Thr Gly Gly Thr Tyr Ala 245 250ttgacaactc ttgcctgagc tttttggtct tgttctgatt tggaaggtga attgagcagg 822tctgctgctg ttggcctctg gagttcattt agttaaagca catgtacact ggtgttggac 882agagcagctt ggcttttcat gtgcccaact acttactact actgcgat 930 18 253 PRT Homosapiens DOMAIN (1)..(11) Cytoplasmic (Potential) 18 Met Gly Gln Cys GlyIle Thr Ser Ser Lys Thr Val Leu Val Phe Leu 1 5 10 15 Asn Leu Ile PheTrp Gly Ala Ala Gly Ile Leu Cys Tyr Val Gly Ala 20 25 30 Tyr Val Phe IleThr Tyr Asp Asp Tyr Asp His Phe Phe Glu Asp Val 35 40 45 Tyr Thr Leu IlePro Ala Val Val Ile Ile Ala Val Gly Ala Leu Leu 50 55 60 Phe Ile Ile GlyLeu Ile Gly Cys Cys Ala Thr Ile Arg Glu Ser Arg 65 70 75 80 Cys Gly LeuAla Thr Phe Val Ile Ile Leu Leu Leu Val Phe Val Thr 85 90 95 Glu Val ValVal Val Val Leu Gly Tyr Val Tyr Arg Ala Lys Val Glu 100 105 110 Asn GluVal Asp Arg Ser Ile Gln Lys Val Tyr Lys Thr Tyr Asn Gly 115 120 125 ThrAsn Pro Asp Ala Ala Ser Arg Ala Ile Asp Tyr Val Gln Arg Gln 130 135 140Leu His Cys Cys Gly Ile His Asn Tyr Ser Asp Trp Glu Asn Thr Asp 145 150155 160 Trp Phe Lys Glu Thr Lys Asn Gln Ser Val Pro Leu Ser Cys Cys Arg165 170 175 Glu Thr Ala Ser Asn Cys Asn Gly Ser Leu Ala His Pro Ser AspLeu 180 185 190 Tyr Ala Glu Gly Cys Glu Ala Leu Val Val Lys Lys Leu GlnGlu Ile 195 200 205 Met Met His Val Ile Trp Ala Ala Leu Ala Phe Ala AlaIle Gln Leu 210 215 220 Leu Gly Met Leu Cys Ala Cys Ile Val Leu Cys ArgArg Ser Arg Asp 225 230 235 240 Pro Ala Tyr Glu Leu Leu Ile Thr Gly GlyThr Tyr Ala 245 250 19 1317 DNA Homo sapiens Gene (1)..(1317) VasoactiveIntestinal Peptide Receptor-2 19 atg cgg acg ctg ctg cct ccc gcg ctg ctgacc tgc tgg ctg ctc gcc 48 Met Arg Thr Leu Leu Pro Pro Ala Leu Leu ThrCys Trp Leu Leu Ala 1 5 10 15 ccc gtg aac agc att cac cca gaa tgc cgattt cat ctg gaa ata cag 96 Pro Val Asn Ser Ile His Pro Glu Cys Arg PheHis Leu Glu Ile Gln 20 25 30 gag gaa gaa aca aaa tgt aca gag ctt ctg aggtct caa aca gaa aaa 144 Glu Glu Glu Thr Lys Cys Thr Glu Leu Leu Arg SerGln Thr Glu Lys 35 40 45 cac aaa gcc tgc agt ggc gtc tgg gac aac atc acgtgc tgg cgg cct 192 His Lys Ala Cys Ser Gly Val Trp Asp Asn Ile Thr CysTrp Arg Pro 50 55 60 gcc aat gtg gga gag acc gtc acg gtg ccc tgc cca aaagtc ttc agc 240 Ala Asn Val Gly Glu Thr Val Thr Val Pro Cys Pro Lys ValPhe Ser 65 70 75 80 aat ttt tac agc aaa gca gga aac ata agc aaa aac tgtacg agt gac 288 Asn Phe Tyr Ser Lys Ala Gly Asn Ile Ser Lys Asn Cys ThrSer Asp 85 90 95 gga tgg tca gag acg ttc cca gat ttc gtc gat gcc tgt ggctac agc 336 Gly Trp Ser Glu Thr Phe Pro Asp Phe Val Asp Ala Cys Gly TyrSer 100 105 110 gac ccg gag gat gag agc aag atc acg ttt tat att ctg gtgaag gcc 384 Asp Pro Glu Asp Glu Ser Lys Ile Thr Phe Tyr Ile Leu Val LysAla 115 120 125 att tat acc ctg ggc tac agt gtc tct ctg atg tct ctt gcaaca gga 432 Ile Tyr Thr Leu Gly Tyr Ser Val Ser Leu Met Ser Leu Ala ThrGly 130 135 140 agc ata att ctg tgc ctc ttc agg aag ctg cac tgc acc aggaat tac 480 Ser Ile Ile Leu Cys Leu Phe Arg Lys Leu His Cys Thr Arg AsnTyr 145 150 155 160 atc cac ctg aac ctg ttc ctg tcc ttc atc ctg aga gccatc tca gtg 528 Ile His Leu Asn Leu Phe Leu Ser Phe Ile Leu Arg Ala IleSer Val 165 170 175 ctg gtc aag gac gac gtt ctc tac tcc agc tct ggc acgttg cac tgc 576 Leu Val Lys Asp Asp Val Leu Tyr Ser Ser Ser Gly Thr LeuHis Cys 180 185 190 cct gac cag cca tcc tcc tgg gtg ggc tgc aag ctg agcctg gtc ttc 624 Pro Asp Gln Pro Ser Ser Trp Val Gly Cys Lys Leu Ser LeuVal Phe 195 200 205 ctg cag tac tgc atc atg gcc aac ttc ttc tgg ctg ctggtg gag ggg 672 Leu Gln Tyr Cys Ile Met Ala Asn Phe Phe Trp Leu Leu ValGlu Gly 210 215 220 ctc tac ctc cac acc ctc ctg gtg gcc atg ctc ccc cctaga agg tgc 720 Leu Tyr Leu His Thr Leu Leu Val Ala Met Leu Pro Pro ArgArg Cys 225 230 235 240 ttc ctg gcc tac ctc ctg atc gga tgg ggc ctc cccacc gtc tgc atc 768 Phe Leu Ala Tyr Leu Leu Ile Gly Trp Gly Leu Pro ThrVal Cys Ile 245 250 255 ggt gca tgg act gcg gcc agg ctc tac tta gaa gacacc ggt tgc tgg 816 Gly Ala Trp Thr Ala Ala Arg Leu Tyr Leu Glu Asp ThrGly Cys Trp 260 265 270 gat aca aac gac cac agt gtg ccc tgg tgg gtc atacga ata ccg att 864 Asp Thr Asn Asp His Ser Val Pro Trp Trp Val Ile ArgIle Pro Ile 275 280 285 tta att tcc atc atc gtc aat ttt gtc ctt ttc attagt att ata cga 912 Leu Ile Ser Ile Ile Val Asn Phe Val Leu Phe Ile SerIle Ile Arg 290 295 300 att ttg ctg cag aag tta aca tcc cca gat gtc ggcggc aac gac cag 960 Ile Leu Leu Gln Lys Leu Thr Ser Pro Asp Val Gly GlyAsn Asp Gln 305 310 315 320 tct cag tac aag agg ctg gcc aag tcc acg ctcctg ctt atc ccg ctg 1008 Ser Gln Tyr Lys Arg Leu Ala Lys Ser Thr Leu LeuLeu Ile Pro Leu 325 330 335 ttc ggc gtc cac tac atg gtg ttt gcc gtg tttccc atc agc atc tcc 1056 Phe Gly Val His Tyr Met Val Phe Ala Val Phe ProIle Ser Ile Ser 340 345 350 tcc aaa tac cag ata ctg ttt gag ctg tgc ctcggg tcg ttc cag ggc 1104 Ser Lys Tyr Gln Ile Leu Phe Glu Leu Cys Leu GlySer Phe Gln Gly 355 360 365 ctg gtg gtg gcc gtc ctc tac tgt ttc ctg aacagt gag gtg cag tgc 1152 Leu Val Val Ala Val Leu Tyr Cys Phe Leu Asn SerGlu Val Gln Cys 370 375 380 gag ctg aag cga aaa tgg cga agc cgg tgc ccgacc ccg tcc gcg agc 1200 Glu Leu Lys Arg Lys Trp Arg Ser Arg Cys Pro ThrPro Ser Ala Ser 385 390 395 400 cgg gat tac agg gtc tgc ggt tcc tcc ttctcc cac aac ggc tcg gag 1248 Arg Asp Tyr Arg Val Cys Gly Ser Ser Phe SerHis Asn Gly Ser Glu 405 410 415 ggc gcc ctg cag ttc cac cgc gcg tcc cgagcc cag tcc ttc ctg caa 1296 Gly Ala Leu Gln Phe His Arg Ala Ser Arg AlaGln Ser Phe Leu Gln 420 425 430 acg gag acc tcg gtc atc tag 1317 Thr GluThr Ser Val Ile 435 20 438 PRT Homo sapiens SIGNAL (1)..(23) Potential20 Met Arg Thr Leu Leu Pro Pro Ala Leu Leu Thr Cys Trp Leu Leu Ala 1 510 15 Pro Val Asn Ser Ile His Pro Glu Cys Arg Phe His Leu Glu Ile Gln 2025 30 Glu Glu Glu Thr Lys Cys Ala Glu Leu Leu Arg Ser Gln Thr Glu Lys 3540 45 His Lys Ala Cys Ser Gly Val Trp Asp Asn Ile Thr Cys Trp Arg Pro 5055 60 Ala Asn Val Gly Glu Thr Val Thr Val Pro Cys Pro Lys Val Phe Ser 6570 75 80 Asn Phe Tyr Ser Lys Ala Gly Asn Ile Ser Lys Asn Cys Thr Ser Asp85 90 95 Gly Trp Ser Glu Thr Phe Pro Asp Phe Val Asp Ala Cys Gly Tyr Ser100 105 110 Asp Pro Glu Asp Glu Ser Lys Ile Thr Phe Tyr Ile Leu Val LysAla 115 120 125 Ile Tyr Thr Leu Gly Tyr Ser Val Ser Leu Met Ser Leu AlaThr Gly 130 135 140 Ser Ile Ile Leu Cys Leu Phe Arg Lys Leu His Cys ThrArg Asn Tyr 145 150 155 160 Ile His Leu Asn Leu Phe Leu Ser Phe Ile LeuArg Ala Ile Ser Val 165 170 175 Leu Val Lys Asp Asp Val Leu Tyr Ser SerSer Gly Thr Leu His Cys 180 185 190 Pro Asp Gln Pro Ser Ser Trp Val GlyCys Lys Leu Ser Leu Val Phe 195 200 205 Leu Gln Tyr Cys Ile Met Ala AsnPhe Phe Trp Leu Leu Val Glu Gly 210 215 220 Leu Tyr Leu His Thr Leu LeuVal Ala Met Leu Pro Pro Arg Arg Cys 225 230 235 240 Phe Leu Ala Tyr LeuLeu Ile Gly Trp Gly Leu Pro Thr Val Cys Ile 245 250 255 Gly Ala Trp ThrAla Ala Arg Leu Tyr Leu Glu Asp Thr Gly Cys Trp 260 265 270 Asp Thr AsnAsp His Ser Val Pro Trp Trp Val Ile Arg Ile Pro Ile 275 280 285 Leu IleSer Ile Ile Val Asn Phe Val Leu Phe Ile Ser Ile Ile Arg 290 295 300 IleLeu Leu Gln Lys Leu Thr Ser Pro Asp Val Gly Gly Asn Asp Gln 305 310 315320 Ser Gln Tyr Lys Arg Leu Ala Lys Ser Thr Leu Leu Leu Ile Pro Leu 325330 335 Phe Gly Val His Tyr Met Val Phe Ala Val Phe Pro Ile Ser Ile Ser340 345 350 Ser Lys Tyr Gln Ile Leu Phe Glu Leu Cys Leu Gly Ser Phe GlnGly 355 360 365 Leu Val Val Ala Val Leu Tyr Cys Phe Leu Asn Ser Glu ValGln Cys 370 375 380 Glu Leu Lys Arg Lys Trp Arg Ser Arg Cys Pro Thr ProSer Ala Ser 385 390 395 400 Arg Asp Tyr Arg Val Cys Gly Ser Ser Phe SerArg Asn Gly Ser Glu 405 410 415 Gly Ala Leu Gln Phe His Arg Gly Ser ArgAla Gln Ser Phe Leu Gln 420 425 430 Thr Glu Thr Ser Val Ile 435 21 889DNA Homo sapiens Gene (1)..(889) Pleiotrophin 21 gtcaaaggca ggatcaggttccccgccttc cagtccaaaa atcccgccaa gagagcccca 60 gagcagagga aaatccaaagtggagagagg ggaagaaaga gaccagtgag tcatccgtcc 120 agaaggcggg gagagcagcagcggcccaag caggagctgc agcgagccgg gtacctggac 180 tcagcggtag caacctcgccccttgcaaca aaggcagact gagcgccaga gaggacgttt 240 ccaactcaaa a atg cag gctcaa cag tac cag cag cag cgt cga aaa ttt 290 Met Gln Ala Gln Gln Tyr GlnGln Gln Arg Arg Lys Phe 1 5 10 gca gct gcc ttc ttg gca ttc att ttc atactg gca gct gtg gat act 338 Ala Ala Ala Phe Leu Ala Phe Ile Phe Ile LeuAla Ala Val Asp Thr 15 20 25 gct gaa gca ggg aag aaa gag aaa cca gaa aaaaaa gtg aag aag tct 386 Ala Glu Ala Gly Lys Lys Glu Lys Pro Glu Lys LysVal Lys Lys Ser 30 35 40 45 gac tgt gga gaa tgg cag tgg agt gtg tgt gtgccc acc agt gga gac 434 Asp Cys Gly Glu Trp Gln Trp Ser Val Cys Val ProThr Ser Gly Asp 50 55 60 tgt ggg ctg ggc aca cgg gag ggc act cgg act ggagct gag tgc aag 482 Cys Gly Leu Gly Thr Arg Glu Gly Thr Arg Thr Gly AlaGlu Cys Lys 65 70 75 caa acc atg aag acc cag aga tgt aag atc ccc tgc aactgg aag aag 530 Gln Thr Met Lys Thr Gln Arg Cys Lys Ile Pro Cys Asn TrpLys Lys 80 85 90 caa ttt ggc gcg gag tgc aaa tac cag ttc cag gcc tgg ggagaa tgt 578 Gln Phe Gly Ala Glu Cys Lys Tyr Gln Phe Gln Ala Trp Gly GluCys 95 100 105 gac ctg aac aca gcc ctg aag acc aga act gga agt ctg aagcga gcc 626 Asp Leu Asn Thr Ala Leu Lys Thr Arg Thr Gly Ser Leu Lys ArgAla 110 115 120 125 ctg cac aat gcc gaa tgc cag aag act gtc acc atc tccaag ccc tgt 674 Leu His Asn Ala Glu Cys Gln Lys Thr Val Thr Ile Ser LysPro Cys 130 135 140 ggc aaa ctg acc aag ccc aaa cct caa gca gaa tct aagaag aag aaa 722 Gly Lys Leu Thr Lys Pro Lys Pro Gln Ala Glu Ser Lys LysLys Lys 145 150 155 aag gaa ggc aag aaa cag gag aag atg ctg gat taaaagatgtcac 768 Lys Glu Gly Lys Lys Gln Glu Lys Met Leu Asp 160 165ctgtggaaca taaaaaggac atcagcaaac aggatcagtt aactattgca tttatatgta 828ccgtaggctt tgtattcaaa aattatctat agctaagtac acaataagca aaaacaaaaa 888 g889 22 168 PRT Homo sapiens SIGNAL (1)..(32) 22 Met Gln Ala Gln Gln TyrGln Gln Gln Arg Arg Lys Phe Ala Ala Ala 1 5 10 15 Phe Leu Ala Phe IlePhe Ile Leu Ala Ala Val Asp Thr Ala Glu Ala 20 25 30 Gly Lys Lys Glu LysPro Glu Lys Lys Val Lys Lys Ser Asp Cys Gly 35 40 45 Glu Trp Gln Trp SerVal Cys Val Pro Thr Ser Gly Asp Cys Gly Leu 50 55 60 Gly Thr Arg Glu GlyThr Arg Thr Gly Ala Glu Cys Lys Gln Thr Met 65 70 75 80 Lys Thr Gln ArgCys Lys Ile Pro Cys Asn Trp Lys Lys Gln Phe Gly 85 90 95 Ala Glu Cys LysTyr Gln Phe Gln Ala Trp Gly Glu Cys Asp Leu Asn 100 105 110 Thr Ala LeuLys Thr Arg Thr Gly Ser Leu Lys Arg Ala Leu His Asn 115 120 125 Ala GluCys Gln Lys Thr Val Thr Ile Ser Lys Pro Cys Gly Lys Leu 130 135 140 ThrLys Pro Lys Pro Gln Ala Glu Ser Lys Lys Lys Lys Lys Glu Gly 145 150 155160 Lys Lys Gln Glu Lys Met Leu Asp 165 23 3143 DNA Homo sapiens Gene(1)..(3143) Osteopontin 23 ggggaagtgt gggagcaggt gggctgggca gtggcagaaacctgatgaca caatctcgcc 60 gcctccctgt gttggtggag gatgtctgca gcagcatttaaattctggga gggcttggtt 120 gtcagcagca gcaggaggag gcagagacag catcgtcgggaccagactcg tctcaggcca 180 gttgcagcct tctcagccaa acgccgacca aggtacagcttcagtttgct actgggttgt 240 gcattcagct gaatttcatg gggaagtcca aattctaaggaaaaaaatgt ggtagtataa 300 aaaggtatca ctgttgtaac ctatgaagat gtcagctattcctttgaaat attttgcagg 360 aaaactcact acc atg aga att gca gtg att tgc ttttgc ctc cta ggc 409 Met Arg Ile Ala Val Ile Cys Phe Cys Leu Leu Gly 1 510 atc acc tgt gcc ata cca gtgagtacag ttgcatctta aagaaaattc 457 Ile ThrCys Ala Ile Pro 15 ctgaaaataa ctgaattgtg tgcttccatg tgctaggaggacattcttgt aatctttctt 517 catcttttct gtttctaag gtt aaa cag gct gat tctgga agt tct gag gaa 569 Val Lys Gln Ala Asp Ser Gly Ser Ser Glu Glu 2025 aag cag gtaagcatct tttatgtttt tatatagtta aatcatttac tcaattatgg 625Lys Gln 30 cgagaggtgc aagaaacgta tttgctgcga tcaaatgagt tcatatttgtaaagcaattt 685 gaaagagtgc ctagcccaca gtaagtgcta cataagagtt tgttaaatgaatctgcaaaa 745 aaaaaaaaaa ttacaaaaag gtacctaagg gtccgggtga ctatatgcttccatcaagac 805 tagtgaagaa tggttgtttt ttccattcat ccctacattt ctttttttaataatgataaa 865 catgcaactt ttttgtag ctt tac aac aaa tac cca gat gct gtggcc aca 916 Leu Tyr Asn Lys Tyr Pro Asp Ala Val Ala Thr 35 40 tgg ctaaac cct gac cca tct cag aag cag aat ctc cta gcc cca cag 964 Trp Leu AsnPro Asp Pro Ser Gln Lys Gln Asn Leu Leu Ala Pro Gln 45 50 55 gtatttttaaacttctcata attaaactac agtgatgaaa gatagccaca ctcaggccat 1024 ttgggctgctcagatgaatc ctgccctgcc tgctggcaaa catgtgctta ggacattgac 1084 tgatctgccatgttggcttc tctctgtgtt aagccatcca cagatgaggc tgaaaaataa 1144 aaactgctttggattaaaaa ggttaacttt tgaataaaaa agctaggcat gtgtgatgcg 1204 cactaacacgtgccattcct tcttcag aat gct gtg tcc tct gaa gaa acc aat 1258 Asn Ala ValSer Ser Glu Glu Thr Asn 60 65 gac ttt aaa caa gag gtaagttctc attttcaatcagaggcccat catgccttga 1313 Asp Phe Lys Gln Glu 70 agagatgaaa gaaggcattgcctggattct cttctgatga aatttcatta gcaagttttc 1373 cagctaattg gcagtctaaaacttgctcat aaataaaaca tgtatttact aaatatcaga 1433 aatactaggt ttcctcggataacctaaaag ccatggtatg tactgtgaat gcaaagattc 1493 tgaaactaaa taaaaagaaagatagtaaaa gactaatgtg ctataaaggc taagggaaaa 1553 taaaaaccca tatattaattttcccggcca tcttaatttt cag acc ctt cca agt 1608 Thr Leu Pro Ser 75 aagtcc aac gaa agc cat gac cac atg gat gat atg gat gat gaa gat 1656 Lys SerAsn Glu Ser His Asp His Met Asp Asp Met Asp Asp Glu Asp 80 85 90 gat gatgac cat gtg gac agc cag gac tcc att gac tcg aac gac tct 1704 Asp Asp AspHis Val Asp Ser Gln Asp Ser Ile Asp Ser Asn Asp Ser 95 100 105 gat gatgta gat gac act gat gat tct cac cag tct gat gag tct cac 1752 Asp Asp ValAsp Asp Thr Asp Asp Ser His Gln Ser Asp Glu Ser His 110 115 120 cat tctgat gaa tct gat gaa ctg gtc act gat ttt ccc acg gac ctg 1800 His Ser AspGlu Ser Asp Glu Leu Val Thr Asp Phe Pro Thr Asp Leu 125 130 135 140 ccagca acc gaa gtt ttc act cca gtt gtc ccc aca gta gac aca tat 1848 Pro AlaThr Glu Val Phe Thr Pro Val Val Pro Thr Val Asp Thr Tyr 145 150 155 gatggc cga ggt gat agt gtg gtt tat gga ctg agg tca aaa tct aag 1896 Asp GlyArg Gly Asp Ser Val Val Tyr Gly Leu Arg Ser Lys Ser Lys 160 165 170 aagttt cgc aga cct gac atc cag gtaaatcctt taacagacac acctgatggt 1950 LysPhe Arg Arg Pro Asp Ile Gln 175 180 tctgactagc gctcaagtct aggaaaccacagtttgcata ttcattcatt cattcatcca 2010 ttcattcatc cattcagcaa gaattcattcatattctact ttatgaccat tgaatacaaa 2070 tctttttctg cttggcggtt tttgtaagtctacataattt ctctctagat ttgattctca 2130 aacacaattc tactttttga aatcctggatcaaagtaaca tgctagtatt atttcagcca 2190 gatttagaca atttttagta taagatgacctaaaagctag agagtggaaa aggattacca 2250 tattcccatc cctagccgtt catataattattcttcattt gtgccgtgat tcag tac 2307 Tyr cct gat gct aca gac gag gac atcacc tca cac atg gaa agc gag gag 2355 Pro Asp Ala Thr Asp Glu Asp Ile ThrSer His Met Glu Ser Glu Glu 185 190 195 ttg aat ggt gca tac aag gcc atcccc gtt gcc cag gac ctg aac gcg 2403 Leu Asn Gly Ala Tyr Lys Ala Ile ProVal Ala Gln Asp Leu Asn Ala 200 205 210 cct tct gat tgg gac agc cgt gggaag gac agt tat gaa acg agt cag 2451 Pro Ser Asp Trp Asp Ser Arg Gly LysAsp Ser Tyr Glu Thr Ser Gln 215 220 225 ctg gat gac cag agt gct gaa acccac agc cac aag cag tcc aga tta 2499 Leu Asp Asp Gln Ser Ala Glu Thr HisSer His Lys Gln Ser Arg Leu 230 235 240 245 tat aag cgg aaa gcc aat gatgag agc aat gag cat tcc gat gtg att 2547 Tyr Lys Arg Lys Ala Asn Asp GluSer Asn Glu His Ser Asp Val Ile 250 255 260 gat agt cag gaa ctt tcc aaagtc agc cgt gaa ttc cac agc cat gaa 2595 Asp Ser Gln Glu Leu Ser Lys ValSer Arg Glu Phe His Ser His Glu 265 270 275 ttt cac agc cat gaa gat atgctg gtt gta gac ccc aaa agt aag gaa 2643 Phe His Ser His Glu Asp Met LeuVal Val Asp Pro Lys Ser Lys Glu 280 285 290 gaa gat aaa cac ctg aaa tttcgt att tct cat gaa tta gat agt gca 2691 Glu Asp Lys His Leu Lys Phe ArgIle Ser His Glu Leu Asp Ser Ala 295 300 305 tct tct gag gtc aat taaaaggagaaaa aatacaattt ctcactttgc 2739 Ser Ser Glu Val Asn 310 atttagtcaaaagaaaaaat gctttatagc aaaatgaaag agaacatgaa atgcttcttt 2799 ctcagtttattggttgaatg tgtatctatt tgagtctgga aataactaat gtgtttgata 2859 attagtttagtttgtggctt catggaaact ccctgtaaac aaaagcttca gggttatgtc 2919 tatgttcattctatagaaga aatgcaaact atcactgtat tttaatattt gttattctct 2979 catgaatagaaatttatgta gaagcaaaca aaatactttt acccacttaa aaagagaata 3039 taacattttatgtcactata atcttttgtt ttttaagtta gtgtatattt tgttgtgatt 3099 atcttttgtggtgtgaataa atcttttatc ttgaatgtaa taag 3143 24 314 PRT Homo sapiensSIGNAL (1)..(16) Potential 24 Met Arg Ile Ala Val Ile Cys Phe Cys LeuLeu Gly Ile Thr Cys Ala 1 5 10 15 Ile Pro Val Lys Gln Ala Asp Ser GlySer Ser Glu Glu Lys Gln Leu 20 25 30 Tyr Asn Lys Tyr Pro Asp Ala Val AlaThr Trp Leu Asn Pro Asp Pro 35 40 45 Ser Gln Lys Gln Asn Leu Leu Ala ProGln Asn Ala Val Ser Ser Glu 50 55 60 Glu Thr Asn Asp Phe Lys Gln Glu ThrLeu Pro Ser Lys Ser Asn Glu 65 70 75 80 Ser His Asp His Met Asp Asp MetAsp Asp Glu Asp Asp Asp Asp His 85 90 95 Val Asp Ser Gln Asp Ser Ile AspSer Asn Asp Ser Asp Asp Val Asp 100 105 110 Asp Thr Asp Asp Ser His GlnSer Asp Glu Ser His His Ser Asp Glu 115 120 125 Ser Asp Glu Leu Val ThrAsp Phe Pro Thr Asp Leu Pro Ala Thr Glu 130 135 140 Val Phe Thr Pro ValVal Pro Thr Val Asp Thr Tyr Asp Gly Arg Gly 145 150 155 160 Asp Ser ValVal Tyr Gly Leu Arg Ser Lys Ser Lys Lys Phe Arg Arg 165 170 175 Pro AspIle Gln Tyr Pro Asp Ala Thr Asp Glu Asp Ile Thr Ser His 180 185 190 MetGlu Ser Glu Glu Leu Asn Gly Ala Tyr Lys Ala Ile Pro Val Ala 195 200 205Gln Asp Leu Asn Ala Pro Ser Asp Trp Asp Ser Arg Gly Lys Asp Ser 210 215220 Tyr Glu Thr Ser Gln Leu Asp Asp Gln Ser Ala Glu Thr His Ser His 225230 235 240 Lys Gln Ser Arg Leu Tyr Lys Arg Lys Ala Asn Asp Glu Ser AsnGlu 245 250 255 His Ser Asp Val Ile Asp Ser Gln Glu Leu Ser Lys Val SerArg Glu 260 265 270 Phe His Ser His Glu Phe His Ser His Glu Asp Met LeuVal Val Asp 275 280 285 Pro Lys Ser Lys Glu Glu Asp Lys His Leu Lys PheArg Ile Ser His 290 295 300 Glu Leu Asp Ser Ala Ser Ser Glu Val Asn 305310 25 259 PRT Homo sapiens Gene (1)..(259) Carbonic Anhydrase domain ofhuman carbonic anhydrase III 25 Ala Lys Glu Trp Gly Tyr Ala Ser His AsnGly Pro Asp His Trp His 1 5 10 15 Glu Leu Phe Pro Asn Ala Lys Gly GluAsn Gln Ser Pro Ile Glu Leu 20 25 30 His Thr Lys Asp Ile Arg His Asp ProSer Leu Gln Pro Trp Ser Val 35 40 45 Ser Tyr Asp Gly Gly Ser Ala Lys ThrIle Leu Asn Asn Gly Lys Thr 50 55 60 Cys Arg Val Val Phe Asp Asp Thr TyrAsp Arg Ser Met Leu Arg Gly 65 70 75 80 Gly Pro Leu Pro Gly Pro Tyr ArgLeu Arg Gln Phe His Leu His Trp 85 90 95 Gly Ser Ser Asp Asp His Gly SerGlu His Thr Val Asp Gly Val Lys 100 105 110 Tyr Ala Ala Glu Leu His LeuVal His Trp Asn Pro Lys Tyr Asn Thr 115 120 125 Phe Lys Glu Ala Leu LysGln Arg Asp Gly Ile Ala Val Ile Gly Ile 130 135 140 Phe Leu Lys Ile GlyHis Glu Asn Gly Glu Phe Gln Ile Phe Leu Asp 145 150 155 160 Ala Leu AspLys Ile Lys Thr Lys Gly Lys Glu Ala Pro Phe Thr Lys 165 170 175 Phe AspPro Ser Cys Leu Phe Pro Ala Cys Arg Asp Tyr Trp Thr Tyr 180 185 190 GlnGly Ser Phe Thr Thr Pro Pro Cys Glu Glu Cys Ile Val Trp Leu 195 200 205Leu Leu Lys Glu Pro Met Thr Val Ser Ser Asp Gln Met Ala Lys Leu 210 215220 Arg Ser Leu Leu Ser Ser Ala Glu Asn Glu Pro Pro Val Pro Leu Val 225230 235 240 Ser Asn Trp Arg Pro Pro Gln Pro Ile Asn Asn Arg Val Val ArgAla 245 250 255 Ser Phe Lys 26 260 PRT Homo sapiens Gene (1)..(260)Carbonic anhydrase domain of human carbonic anhydrase I 26 Ala Ser ProAsp Trp Gly Tyr Asp Asp Lys Asn Gly Pro Glu Gln Trp 1 5 10 15 Ser LysLeu Tyr Pro Ile Ala Asn Gly Asn Asn Gln Ser Pro Val Asp 20 25 30 Ile LysThr Ser Glu Thr Lys His Asp Thr Ser Leu Lys Pro Ile Ser 35 40 45 Val SerTyr Asn Pro Ala Thr Ala Lys Glu Ile Ile Asn Val Gly His 50 55 60 Ser PheHis Val Asn Phe Glu Asp Asn Asp Asn Arg Ser Val Leu Lys 65 70 75 80 GlyGly Pro Phe Ser Asp Ser Tyr Arg Leu Phe Gln Phe His Phe His 85 90 95 TrpGly Ser Thr Asn Glu His Gly Ser Glu His Thr Val Asp Gly Val 100 105 110Lys Tyr Ser Ala Glu Leu His Val Ala His Trp Asn Ser Ala Lys Tyr 115 120125 Ser Ser Leu Ala Glu Ala Ala Ser Lys Ala Asp Gly Leu Ala Val Ile 130135 140 Gly Val Leu Met Lys Val Gly Glu Ala Asn Pro Lys Leu Gln Lys Val145 150 155 160 Leu Asp Ala Leu Gln Ala Ile Lys Thr Lys Gly Lys Arg AlaPro Phe 165 170 175 Thr Asn Phe Asp Pro Ser Thr Leu Leu Pro Ser Ser LeuAsp Phe Trp 180 185 190 Thr Tyr Pro Gly Ser Leu Thr His Pro Pro Leu TyrGlu Ser Val Thr 195 200 205 Trp Ile Ile Cys Lys Glu Ser Ile Ser Val SerSer Glu Gln Leu Ala 210 215 220 Gln Phe Arg Ser Leu Leu Ser Asn Val GluGly Asp Asn Ala Val Pro 225 230 235 240 Met Gln His Asn Asn Arg Pro ThrGln Pro Leu Lys Gly Arg Thr Val 245 250 255 Arg Ala Ser Phe 260 27 337PRT Homo sapiens Gene (1)..(337) Carbonic anhydrase domain of humancarbonic anhydrase VIX 27 Met Leu Phe Ser Ala Leu Leu Leu Glu Val IleTrp Ile Leu Ala Ala 1 5 10 15 Asp Gly Gly Gln His Trp Thr Tyr Glu GlyPro His Gly Gln Asp His 20 25 30 Trp Pro Ala Ser Tyr Pro Glu Cys Gly AsnAsn Ala Gln Ser Pro Ile 35 40 45 Asp Ile Gln Thr Asp Ser Val Thr Phe AspPro Asp Leu Pro Ala Leu 50 55 60 Gln Pro His Gly Tyr Asp Gln Pro Gly ThrGlu Pro Leu Asp Leu His 65 70 75 80 Asn Asn Gly His Thr Val Gln Leu SerLeu Pro Ser Thr Leu Tyr Leu 85 90 95 Gly Gly Leu Pro Arg Lys Tyr Val AlaAla Gln Leu His Leu His Trp 100 105 110 Gly Gln Lys Gly Ser Pro Gly GlySer Glu His Gln Ile Asn Ser Glu 115 120 125 Ala Thr Phe Ala Glu Leu HisIle Val His Tyr Asp Ser Asp Ser Tyr 130 135 140 Asp Ser Leu Ser Glu AlaAla Glu Arg Pro Gln Gly Leu Ala Val Leu 145 150 155 160 Gly Ile Leu IleGlu Val Gly Glu Thr Lys Asn Ile Ala Tyr Glu His 165 170 175 Ile Leu SerHis Leu His Glu Val Arg His Lys Asp Gln Lys Thr Ser 180 185 190 Val ProPro Phe Asn Leu Arg Glu Leu Leu Pro Lys Gln Leu Gly Gln 195 200 205 TyrPhe Arg Tyr Asn Gly Ser Leu Thr Thr Pro Pro Cys Tyr Gln Ser 210 215 220Val Leu Trp Thr Val Phe Tyr Arg Arg Ser Gln Ile Ser Met Glu Gln 225 230235 240 Leu Glu Lys Leu Gln Gly Thr Leu Phe Ser Thr Glu Glu Glu Pro Ser245 250 255 Lys Leu Leu Val Gln Asn Tyr Arg Ala Leu Gln Pro Leu Asn GlnArg 260 265 270 Met Val Phe Ala Ser Phe Ile Gln Ala Gly Ser Ser Tyr ThrThr Gly 275 280 285 Glu Met Leu Ser Leu Gly Val Gly Ile Leu Val Gly CysLeu Cys Leu 290 295 300 Leu Leu Ala Val Tyr Phe Ile Ala Arg Lys Ile ArgLys Lys Arg Leu 305 310 315 320 Glu Asn Arg Lys Ser Val Val Phe Thr SerAla Gln Ala Thr Thr Glu 325 330 335 Ala 28 22 DNA Artificial sequencePrimer 28 cagcagttgg atggaagagg ac 22 29 22 DNA Artificial sequencePrimer 29 cactgagatt ctggcactat tc 22 30 21 DNA Artificial sequencePrimer 30 aacaattcca gggtctcact c 21 31 21 DNA Artificial sequencePrimer 31 ttgactggct caggagtata g 21 32 21 DNA Artificial sequencePrimer 32 ctgataatga gggctcccaa c 21 33 24 DNA Artificial sequencePrimer 33 ctctgcactt cctggtaaaa ctct 24 34 22 DNA Artificial sequencePrimer 34 cagcagttgg atggaagagg ac 22 35 24 DNA Artificial sequencePrimer 35 ctctgcactt cctggtaaaa ctct 24

We claim:
 1. A method to treat a brain tumor comprising administering atherapeutic amount of a composition comprising: a compound of thegeneral formula α(VIPR2), wherein α(VIPR2) is one or more moieties whichspecifically binds to a human vasoactive intestinal peptide receptor-2,wherein the binding of α(VIPR2) alters the function of the vasoactiveintestinal peptide receptor-2, and a pharmaceutically acceptablecarrier.
 2. The method of claim 1 wherein the therapeutic composition isadministered by intrathecal administration.
 3. The method of claim 1wherein the therapeutic composition is administered by intravascularadministration.
 4. The method of claim 1 wherein the brain tumor is aglioblastoma.
 5. The method of claim 1 wherein α(VIPR2) is selected fromthe group consisting of an antibody and an antibody fragment.
 6. Themethod of claim 1 wherein the composition is administered as a singledose.
 7. The method of claim 1 wherein the composition is administeredas a series of doses over a period of time.
 8. The method of claim 1wherein α(VIPR2) further comprises attached thereto one or morecytotoxic moieties C.
 9. The method of claim 8 wherein the therapeuticcomposition is administered by intrathecal administration.
 10. Themethod of claim 8 wherein the therapeutic composition is administered byintravascular administration.
 11. The method of claim 8 wherein thebrain tumor is a glioblastoma.
 12. The method of claim 8 whereinα(VIPR2) is selected from the group consisting of an antibody and anantibody fragment.
 13. The method of claim 8 wherein C is a radioactivemoiety.
 14. The method of claim 8 wherein C is a chemotoxic moiety. 15.The method of claim 8 wherein C is a toxin protein moiety.
 16. Themethod of claim 8 wherein the composition is administered as a singledose.
 17. The method of claim 8 wherein the composition is administeredas a series of doses over a period of time.